Dendritic Cell Populations that are Enriched for Cells that Bear the BDCA-1 Antigen

ABSTRACT

The invention provides antigen-binding fragments specific for dendritic cells and effective in treatment and/or diagnosing a variety of disorders. Methods of use are also provided as are methods for screening for additional such antigen-binding fragments and the products obtained thereby.

CROSS-REFERENCES TO RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.11/393,155 (pending), filed Mar. 29, 2006, which claims priority to U.S.patent application Ser. No. 09/714,712, filed Nov. 15, 2000; and claimsthe benefit of U.S. Provisional Application Ser. No. 60/197,205, filedApr. 13, 2000; U.S. Provisional Application Ser. No. 60/196,824, filedApr. 11, 2000; U.S. Provisional Application Ser. No. 60/180,775, filedFeb. 7, 2000; U.S. Provisional Application Ser. No. 60/179,003, filedJan. 28, 2000; U.S. Provisional Application Ser. No. 60/167,076, filedNov. 23, 1999; and U.S. Provisional Application Ser. No. 60/165,555,filed Nov. 15, 1999.

The above listed applications are hereby incorporated herein in theirentirety for all purposes.

TECHNICAL FIELD

The present invention relates to antibodies and derivatives thereofspecific for subpopulations of dendritic cells (DCs). Compositions andmethods of use thereof are also provided including isolation andpurification of DCs and subpopulations thereof and antibody- orligand-mediated immunotherapy. The invention also provides substantiallyisolated DC subpopulations. Methods of use thereof are also providedincluding DC-based immunotherapy, characterization of various diseasesand in vivo numeric DC expansion for instance with flt3-Ligand.

BACKGROUND OF THE INVENTION

The hematopoietic development of dendritic cells (DCs), potent antigenpresenting cells (APCs) is distinct and may follow several precursorpathways some closely linked to monocytes. DCs may be derived from alymphoid precursor. Thomas et al. (1993) J. Immunol. 150:821-834. Likein blood, there may be three distinct subsets of DCs present in thethymus: 1) plasmacytoid CD4+CD11c− DCs; 2) CD4+CD11c+DCs and 3)interdigitating DCs. It has been proposed that thymic DCs and T cellsarise from a common stem cell. Thomas et al. (1996) Stem Cells14:196-206.

Generation of large numbers of DCs for potential clinical use hasrecently been accomplished through the in vitro culturing of progenitorswith cytokines. Various strategies have been adopted to introduceantigens into dendritic cells so that they may be more effectivelypresented to T cells in the context of costimulation. It has also beenshown that dendritic cells can influence the T cell response to antigento follow either a humoral or systemic pathway.

T cells are unable to respond to unprocessed proteins, rather, theyrequire accessory cells to present antigen as peptide epitopes displayedon the cell surface in conjunction with MHC molecules. Antigensgenerated endogenously in the cell cytoplasm are typically presented inthe Class I pathway and stimulate cytotoxic T lymphocyte (CTL) reactionswhile exogenous protein is process in MHC Class II compartments andinduce helper (CD4) T cell responses. The stimulation of naïve T cellsrequires the presence of costimulatory molecules that act as secondarysignals in the activation of primary immunity. APCs such as B cells andmacrophages are typically incapable of inducing primary responses. Incontrast, dendritic cells drive their potency from the constitutiveunregulated expression of costimulatory, adhesion and MHC Class I and IImolecules essential for the initiation of effective cellular immunity.For review see, Avigan (1999) Blood Rev. 13:51-64.

DCs are APC that are essential for initiation of primary immuneresponses and the development of tolerance. DCs express MHC, necessaryfor stimulation of naïve T cell populations. The hematopoieticdevelopment of DCs is distinct and may follow several precursorpathways, some of which are closely linked to monocytes. See, forreview, Avigan (1999) Blood Rev. 13:51-64. Different DC subsets havedistinct developmental pathways. The emerging concept is that one DCsubset has regulatory functions that may contribute to the induction oftolerance to self-antigens. Austyn (1998) Curr. Opin. Hematol. 5:3-15.Conversely, DCs, or a subset thereof, may also be involved in theinduction of immune responses to self-proteins. It is thought thatcertain autoimmune responses may be due to microenvironmental tissueinjury followed by local DC activation and subsequent interaction with Tcells to initiate an immune response. Ibrahim et al. (1995) Immunol.Today 16:181-186.

The ability of DCs to initiate T cell responses is being used in DCcancer vaccines. Hart et al. (1999) Sem. Hematol. 36:21-25. Forinstance, DCs are generated in vitro from CD34⁺ cells or monocytes,pulsed with tumor-derived peptides or proteins and returned to thepatient to act as APCs in cancer-specific T cell induction. Brugger etal. (1999) Ann. N.Y. Acad. Sci. 872:363-371. Animal models havedemonstrated that DC tumor vaccines reverse T cell anergy and result insubsequent tumor rejection. Avigan (1999); see also, Tarte et al. (1999)Leukemia 13:653-663; Colaco (1999) Molec. Med. Today 5:14-17; Timmermanet al. (1999) Ann. Rev. Med. 50:507-529; Hart et al. (1999) Semin.Hematol. 36:21-25; Thurnher et al. (1998) Urol. Int. 61:67-71; andHermans et al. (1998) N.Z. Med. J. 111:111-113. One approach has been toincrease DCs in vivo by administration of flt-Ligand. This has theeffect of compensating for VEGF-induced DC suppression. Ohm et al.(1999) J. Immunol. 163:3260-3268. DCs have been proposed for use asadjuvants in vaccination and in recombinant vaccines. Fernandez et al.(1998) Cyto. Cell. Mol. Ther. 4:53-65; and Gilboa et al. (1998) CancerImmunol. Immunother. 46:82-87. DC have also been proposed for use inenhancing immunity after stem cell transplantation. Brugger et al.(1999) Ann. NY Acad. Sci. 363-371. DCs play a number of potential rolesin immunology. For instance, DCs are involved in human immunodeficiencyvirus (HIV) infection. Zoeteweij et al. (1998) J. Biomed. Sci.5:253-259. DCs have also been proposed as suitable for use in HIVtherapy. Weissman et al. (1997) Clin. Microbiol. Rev. 10:358-367.

Additional immunologic functions are related to DCs such as differentialinduction of Th1 or Th2 responses, autoimmune reactions and allergies.Rissoan et al. (1999) Science 283:1183-1186; Hermans et al. (1998) NZMed. J. 111:111-113; and De Palma et al. (1999) J. Immunol.162:1982-1987.

Increased levels of circulating IFN-α and of IFN-α inducing factor(something like a complex of anti-DNA antibody and DNA) are found in SLEpatients and correlate to disease activity. Furthermore, patients withnon-autoimmune disorders treated with IFN-α frequently developautoantibodies and occasionally SLE. Several papers from Ronnblom et al.(1999) Clin. Exp. Immunol. 115: 196-202; (1999) J. Immunol. 163:6306-6313; and (2000) J. Immunol. 165: 3519-3526) show that IFN-αinducing factors derived from patients induce secretion of IFN-α in PBMCfrom healthy donors and they selectively activate natural IFN-αproducing cells (NIPC=plasmacytoid DC).

Studies on DC's in blood have been hampered by scarcity of the cells andthe relative lack of DC-specific cell surface markers. Methods for DCisolation are based on either maturational change after a short cultureperiod, like the acquisition of low buoyant density or the expression ofDC activation/maturation antigens (CD83, CMRF-44 and CMRF-56). Young etal. (1988) Cell Immunol. 111:167; Van Voorhis et al. (1982) J. Exp. Med.155:1172; Zhou et al. (1995) J. Immunol. 154:3821-3835; Fearnley et al.(1997) Blood 89:3708-3716; Mannering et al. (1988) J. Immunol. Met.219:69-83; Hock et al. (1999) Tiss. Antigens 53:320-334; and Hock et al.Immunol. 83:573-581.

Functional CD1a⁺ DCs are typically generated ex vivo from monocytes andfrom CD34⁺ hematopoietic progenitor cells. Bender et al. (1996) J.Immunol. Met. 196:121-135; Pickl et al. (1996) J. Immunol.157:3850-3859; Romani et al. (1994) J. Exp. Med. 180:83-93; Sallusto etal. (1994) J. Exp. Med. 179:1109-1118; Caux et al. (1992) Nature360:258-261; Mackensen et al. (1995) Blood 86:2699-2707; Szabolcs et al.(1995) J. Immunol. 154:5851-5861; Herbst et al. (1996) Blood88:2541-2548; de Wynter et al. (1998) Stem Cells 16:387-396; Strunk etal. (1996) Blood 87:1292-1302 U.S. Pat. Nos. 6,010,905; and 6,004,807.It is not known if DCs generated in vitro from monocytes andhematopoietic progenitor cells retain or obtain all of thecharacteristics of in vivo DCs.

In addition, several attempts to generate mAb specific for human DC havefailed, yielding only mAb that bind antigens expressed by both DC andother leukocytes. Human-DC share a large number of immunogenic cellsurface structures with other blood cells, including HLA molecules,CD18, CD29, CD31, CD43, CD44, CD45, CD54, and CD58. These antigens maydominate the immune response to injected DC to a level where B cellswith specificity for DC-specific antigens are not at all or only veryrarely represented among B cells that have the capability to fuse withmyeloma cells.

Many investigators have tried to overcome this problem by injectingadult mice with non-DC and cyclophosphamide, in order to ablate B cellswith specificity for shared antigens, or by injecting neonatal mice withnon-DC, in order to tolerize B cells with specificity for sharedantigens. O'Doherty et al. (1993) Adv. Exp. Med. Biol. 329:165-172; andYamaguchi et al. (1995) J. Immunol. Met. 181:115-124.

A mAb designated CMRF44 has been used to monitor DCs in stem celltransplant patients. Fearnley et al. (1999) Blood 93:728-736. TheseCMRF44⁺ cells were proposed to be suitable for use in initiating,maintaining and directing immune responses. Fearnley et al. (1997). DCshave been isolated most often by using a combination of cell surfacemarkers. For instance, U.S. Pat. No. 5,972,627 describes “hematopoieticcells enriched for human hematopoietic dendritic progenitor cells” ashaving “at least 80% expressing CD34, CD45RA, and CD10 but not CD19,CD2, CD3, CD4, CD8, CD20, CD14, CD15, CD16 CD56 and glycophorin.”

Isolation of DCs from blood relies on a multitude of immunophenotypiccriteria, like the absence of a panel of leukocyte lineage(lin)-specific antigens (e.g. CD3, CD14, CD19 and CD56) and the presenceof HLA-DR, CD4 or CD33. Romani et al. (1996) J. Immunol. Met.196:137-151; Thomas et al. (1993) J. Immunol. 150:821-834; Thomas et al.(1994) J. Immunol. 153:4016-4028; O'Doherty et al. (1994) Immunol.82:487-493; O'Doherty et al. (1993) J. Exp. Med. 178:1067-1076; Nijmanet al. (1995) J. Exp. Med. 182:163-174; Ferbas et al. (1994) J. Immunol.152:4649-4662; Heufler et al. (1996) Eur. J. Immunol. 26:659-668; Ito etal. (1999) J. Immunol. 163:1409-1419; Cella et al. (1999) Nature Med.5:919-923; Robinson et al. (1999) Eur. J. Immunol. 29:2769-2778; Olweuset al. (1997) Proc. Natl. Acad. Sci. USA 94:12551-12556; Robert et al.(1999) J. Exp. Med. 189:627-636; and Kohrgruber et al. (1999) J.Immunol. 163:3250-3259.

From analyses of DC isolated from non-cultured blood it became evidentthat blood DC are not a homogeneous cell population but a mixture of atleast two populations. Thomas et al. (1994); O'Doherty et al. (1994);Ito et al. (1999); Cella et al. (1999); Robinson et al. (1999); Olweuset al. (1997); Kohrgruber et al. (1999); Strobl et al. (1998) J.Immunol. 161:740-748; and Rissoan et al. (1999) Science 283:1183-1186.The first blood DC subpopulation is CD 123^(bright) CD 11c⁻ DC, whichpossesses a plasmacytoid morphology and potent T cell stimulatoryfunction. The second blood DC subpopulation is CD123^(dim) CDC^(bright), which is rather monocytoid in appearance, expresses CD45ROand spontaneously develops into typical mature DCs even when culturedwithout any exogenous cytokines. Plasmacytoid CD123^(bright) CD11c⁻ DCdisplay some features, like the expression of the pre-T cell receptor αchain, which indicate that they may arise from lymphoid precursors.Strobl et al. (1998); Rissoan et al. (1999); and Bruno et al. (1997) J.Exp. Med. 185:875-884. CD123^(dim) CD11c^(bright) DC display all thecriteria of myeloid DCs. O'Doherty et al. (1994); and Ito et al. (1999).Robinson et al. (1999); Kohrgruber et al. (1999); and Strobl et al.(1998). DCs resembling plasmacytoid CD123^(bright)CD11c⁻ DC have beendetected in the T cell-rich areas of lymphoid tissue and were initiallyerroneously designated plasmacytoid T cells or plasmacytoid monocytesdue to their morphology and phenotype. Grouard et al. (1997) J. Exp.Med. 185:1101-1111; Lennert et al. (1975) Lancet 1:1031-1032; Lennert etal. (1984) in Leukocyte Typing. Human Leukocyte differentiation antigensdetected by monoclonal antibodies. Bernard et al. eds. Springer-Verlag,Berlin; and Facchetti et al. (1988) Am. J. Pathol. 133:15. DCsresembling CD123^(dim)CD11c^(bright) blood DC have been found in thedark and light zone of germinal centers. Grouard (1996) Nature384:364-367.

Splice Variants

Estimates of the total number of expressed genes range from 40,000 tomore than 150,000. This number is not an accurate reflection of thenumber of proteins encoded since, in many cases, more than one splicevariant from the mRNAs (transcriptome) produced from these genes.Estimates again vary, but perhaps as many as 500,000 different mRNAs areproduced in the human. It is estimated that at least 30% of the humangenes have several splice variants. Mironov et al. (1999) GenomeResearch 9:1288-1293). These numbers are believed by some to beconservative. Similar numbers are believed to be true for mouse and ratand alternative splicing occurs also in lower organisms, such asDrosophila melantogaster and Caenorhabditis elegans. Proteins translatedfrom different splice variants can have significantly differentfunctions, as evidenced by a growing number of research papers.Different splice variants may be expressed in different tissues,different developmental stages and different disease states.

C-Type Lectins

C-type lectins are a family of glycoproteins that exhibit amino acidsequence similarities in their carbohydrate recognition domains (CRD)and that bind to selected carbohydrates in a Ca²⁺-dependent manner.C-type lectins have been subdivided into four categories (Vasta et al.,1994; and Spiess 1990). The first group comprises type IImembrane-integrated proteins, such as asialoglycoprotein receptors,macrophage galactose and N-acetyl glucosamine (GlcNac)-specific lectin,and CD23 (Fc_(ε)RII). Many members in this group exhibit specificity forgalactose/fucose, galactosamine/GalNac or GlcNac residues. The secondgroup includes cartilage and fibroblast proteoglycan core proteins. Thethird group includes the so-called “collectins” such as serummannose-binding proteins, pulmonary surfactant protein SP-A, andconglutinin. The fourth group includes certain adhesion molecules knownas LEC-CAMs (e.g., Mel-14, GMP-140; and ELAM-1).

C-type lectins are known to function as agglutinins, opsonins,complement activators, and cell-associated recognition molecules (Vastaet al. 1994; Spiess 1990; and Kery 1991). For instance, macrophagemannose receptors serve a scavenger function (Shepherd et al., 1990), aswell as mediating the uptake of pathogenic organisms, includingPneumocystis carinii (Ezekowitz et al. 1991) and Candida albicans(Ezekowitz et al. 1990). Serum mannose-binding protein mimics Clq in itscapacity to activate complement through the classical pathway. Geneticmutations in this lectin predispose for severe recurrent infections,diarrhea, and failure to thrive (Reid et al. 1994). Thus, C-type lectinsexhibit diverse functions with biological significance.

Carbohydrate moieties do not necessarily serve as “natural” ligands forC-type lectins. For example, CD23 (FC_(ε)RII), which belongs to theC-type lectin family as verified by its binding of Gal-Gal-Nac(Kijimoto-Ochiai et al. 1994) and by its CRD sequence, is now known torecognize IgE in a carbohydrate-independent manner; an enzymaticallydeglycosylated form of IgE as well as recombinant (non-glycosylated) IgEproduced in E. coli both bind to CD23 (Vercelli et al. 1989). Thus, someC-type lectins recognize polypeptide sequences in their natural ligands.

Several C-type lectins have been identified on the surface of DCs.First, Jiang et al. cloned the protein recognized by the NLDC-145 mAb,one of the most widely used mAb against murine DC (Jiang et al., 1995).This protein, now termed DEC-205, was found to be a new member of theC-type lectin family, one that contains ten distinct CRD. Second,Sallusto et al. reported that human DC express macrophage mannosereceptors (MMR), which also contain multiple-CRD (Sallusto et al.,1995). Both receptors have been proposed to mediate endocytosis ofglycosylated molecules by DC, based on the observations that: a)polyclonal rabbit antibodies against DEC-205 not only bound to DEC-205on DC surfaces, but were subsequently internalized; b) these DCactivated effectively a T cell line reactive to rabbit IgG; and c)internalization of FITC-dextran by DC was blocked effectively withmannan, a mannose receptor competitor (Jiang et al. 1995; and Sallustoet al. 1995). With respect to cell type specificity, DEC-205 is nowknown to be also expressed, albeit at lower levels, by B cells andepithelial cells in thymus, intestine, and lung (Witmer-Pack et al:1995; and Inaba et al. 1995) and MMR is also expressed even moreabundantly by macrophages (Stahl 1992). Other have also been found on DCsurfaces, these include DCIR, MDL-1, NURPIA, Dectin-1, Dectin-2, CLEC-1,CLEC-2, Langerin; and DC-sign.

Allergies

Allergic responses, including those of allergic asthma and allergicrhinitis, are characterized by an early phase response, which occurswithin seconds to minutes of allergen exposure and is characterized byinfiltration of eosinophils into the site of allergen exposure.Specifically, during the early phase of the allergic response,activation of Th2-type lymphocytes stimulates the production ofantigen-specific IgE antibodies, which in turn triggers the release ofhistamine and other mediators of inflammation from mast cells andbasophils. During the late phase response, IL-4 and IL-5 production byCD4+ Th2 cells is elevated. These cytokines appear to play a significantrole in recruiting eosinophils into the site of allergen exposure, wheretissue damage and dysfunction result.

Currently, antigen immunotherapy for allergic disorders involves thesubcutaneous injection of small, but gradually, increasing amounts, ofantigen in a process called desensitization therapy. Antigenimmunotherapy is merely palliative and, at present, not curative. Weber(1997) JAMA 278:1881-1887; Stevens (1998) Acta Clinica Beligica53:66-72; and Canadian Society of Allergy and Clinical Immunology (1995)Can. Med. Assoc. J. 152:1413-1419.

Many patients who begin the therapy do not complete the regimen, and ifinjections are missed for over a week, the patient must begin the entiretreatment regimen again. A variety of antigens have been identified andproduced by recombinant means. For reviews, see Baldo et al. (1989)Allergy 44:81-97; Baldo (1991) Curr. Opin. Immunol. 3:841-850; Blaser(1994) Ther. Umsch 51:19-23; and Valenta et al. (1996) Adv. Exp. Med.Bio. 409:185-196.

Antigen immunotherapy treatments present the risk of inducingpotentially lethal IgE-mediated anaphylaxis and do not address thecytokine-mediated events of the allergic late phase response. Thistherapy has been described as “having the potential for misadventure.”Weber (1997). Another significant problem with antigen immunotherapy isthat the risk of adverse reactions, especially anaphylaxis,significantly reduces the dosage of antigen both with respect to theamount given per administration and the amount given over a period oftime. Thus, traditional allergy immunotherapy is protracted and thustime-consuming, inconvenient, and expensive.

An alternative approach for treatment of IgE-associated disorders suchas allergies involves administration of compounds that inhibit histaminerelease. Many such drugs are available as over-the-counter remedies.Other drugs include an anti-IgE binding antibody. However, a drawback ofthis approach is that it merely masks the symptoms, while not providingany kind of permanent cure or protection.

BRIEF DESCRIPTION OF THE INVENTION

The invention relates to methods of enriching for hematopoietic cellpopulations enriched in DCs and subsets thereof. Compositions enrichedfor the cells and populations of cells obtained therefrom are alsoprovided by the invention. Methods of making genetically modified DCsare also provided. Compositions of genetically modified DCs are alsoprovided. Methods of use of the cells are also included. Antigen-bindingfragments specific for BDCA-2 and BDCA-3 and the antigens recognizedthereby are also provided.

The invention encompasses antigen-binding fragments specific for asubset of DCs specifically recognized by an antibody designated AC144,AD5-1311, AD5-20E5, AD5-17F6, AD5-4B8, AD5-5E8, AD5-14H12 or AD5-8E7.The invention encompasses antigen-binding fragments specific for anepitope of an antigen designated BDCA-2 (SEQ ID NO:2). The inventionencompasses antigen-binding fragments Specific for an epitope of anantigen designated BDCA-3.

The invention encompasses a substantially isolated or concentrated DCpopulation or subpopulation specifically recognized by anantigen-binding fragment of the invention. These antigen-bindingfragments can be any one of AC144, AD5-1311, AD5-20E5, AD5-17F6,AD5-4B8, AD5-5E8, AD5-14H12 or AD5-8E7 or antigen-binding fragmentsspecific for BDCA-1, BDCA-2, BDCA-3 or BDCA-4. Antigen-binding fragmentsrecognizing neuropilin-1 also recognize BDCA-4 and are suitable for useherein.

The invention further encompasses populations or subpopulations of DCswherein substantially all of the cells express or are isolated,concentrated or enumerated on the basis of expression of at least one ofBDCA-1, BDCA-2, BDCA-3 and BDCA-4. These cells can be suspended in anyphysiologically acceptable excipient. Preferably, the excipient ispharmacologically acceptable.

The invention further encompasses methods for obtaining compositions ofhematopoietic cells enriched for DCs by separating a mixture of humanhematopoietic cells into a fraction wherein at least 80% of the cells inthe fraction are BDCA-1⁺.

The invention further encompasses methods for obtaining compositions ofhematopoietic cells enriched for DCs by separating a mixture of humanhematopoietic cells into a fraction wherein at least 80% of the cells inthe fraction are BDCA-2⁺.

The invention further encompasses methods for obtaining compositions ofhematopoietic cells enriched for DCs by separating a mixture of humanhematopoietic cells into a fraction wherein at least 80% of the cells inthe fraction are BDCA-3⁺.

The invention further encompasses methods for obtaining compositions ofhematopoietic cells enriched for DCs by separating a mixture of humanhematopoietic cells into a fraction wherein at least 80% of the cells inthe fraction are BDCA-4⁺.

The invention further encompasses methods for isolating a substantiallypure subset of DCs by a) obtaining a mixture of human hematopoieticcells; and b) substantially isolating cells from the mixturespecifically recognized by an antigen-binding fragment specific for theantigen designated BDCA-2.

The invention further encompasses methods for isolating a substantiallypure subset of DCs by a) obtaining a mixture of human hematopoieticcells; and b) substantially isolating cells from the mixturespecifically recognized by an antigen-binding fragment specific for theantigen designated BDCA-3.

The invention further encompasses methods for isolating a substantiallypure subset of DCs by a) obtaining a mixture of human hematopoieticcells; and b) substantially isolating cells from the mixturespecifically recognized by an antigen-binding fragment specific for theantigen designated BDCA-4.

The invention further encompasses methods for enumerating DCs by: a)obtaining a mixture of cells; and b) labeling the cells with anantigen-binding fragment specific for any one or more of the antigensBDCA-1, BDCA-2, BDCA-3, and BDCA-4.

The invention further encompasses methods of modulating the immunecapacity of DCs by: isolating a substantially pure population orsubpopulation of DCs; and modulating the calcium mobilization of theDCs.

The invention further encompasses methods of screening for test agentsfor the presence of pharmaceutically effective agents by isolating asubstantially pure population or subpopulation of DCs with anantigen-binding fragment specific for any one or more of the antigensBDCA-1, BDCA-2, BDCA-3, and BDCA-4; screening the isolated cells withtest agents; monitoring the response of the cells to the agents;comparing the response of the cells to the agents to cells exposed to acontrol agent; and determining whether the test agent-modulated any oneimmunologic properties of the isolated cell.

The invention further encompasses methods of modulating an immunologicproperty of DCs by altering the ability of the DC to mobilize calcium.

The invention further encompasses immunogenic and immunomodulatingcompositions of DCs preferably in a physiologically acceptableexcipient.

The invention further encompasses methods of treating a physiologiccondition by administering to a subject in need thereof an effectiveamount of immunogenic or immunomodulating compositions of DCs.

The invention further encompasses methods of producing DC cytokines byisolating a substantially pure population or subpopulation of DCs withan antigen-binding fragment specific for any one or more of BDCA-1,BDCA-2, BDCA-3, and BDCA-4; and isolating cytokines from the cells orcellular products or supernatants.

The invention further encompasses methods of modulating DC cytokineproduction by isolating a substantially pure population or subpopulationof DCs with an antigen-binding fragment specific for any one or more ofBDCA-1, BDCA-2, BDCA-3, and BDCA-4; and treating the cells with agentsthat modulate DC cytokine production.

The invention further encompasses methods of modulating in vivo DCcytokine production by administering to a subject in need thereof aneffective amount of an agent that modulates DC cytokine production.

The invention further encompasses methods of generating antibodiesspecific for an antigen by administering to a subject in need thereof aneffective amount of a substantially pure population or subpopulation ofDCs loaded with the antigen and isolated with an antigen-bindingfragment specific for any one or more of BDCA-1, BDCA-2, BDCA-3, andBDCA-4 wherein the DCs are modulated to induce a Th2 response.

The invention further encompasses methods of generating a T cell orhumoral immune response specific for an antigen by administering to asubject in need thereof an effective amount of a substantially purepopulation or subpopulation of DCs loaded with the antigen and isolatedwith an antigen-binding fragment specific for any one or more of BDCA-1,BDCA-2, BDCA-3, and BDCA-4 wherein the cells are modulated to induce aTh1 response.

The invention further encompasses polypeptides prepared by expressing,in a recombinant host cell, the polypeptides and purifying the expressedpolypeptide away from total recombinant host cell components, whereinthe polypeptide contains about 5 contiguous amino acid residues from SEQID NO:2.

The invention further encompasses of purified polypeptides andcompositions thereof, wherein the polypeptide contains about 5contiguous amino acid residues from SEQ ID NO:2.

The invention further encompasses fusion proteins of a polypeptide aminoacid sequence linked to a polypeptide amino acid sequence that is notSEQ ID NO: 2, wherein the amino acid sequence contains about 5contiguous amino acid residues from SEQ ID NO:2.

The invention further encompasses polypeptides containing at least onesplice variant of BDCA-2.

The invention further encompasses a polynucleotide or a complementthereof encoding at least 5 contiguous amino acid residues of BDCA-2, asplice variant or a fragment thereof.

The invention further encompasses recombinant host cells containing apolynucleotide or a complement thereof encoding at least 5 contiguousamino acid residues of BDCA-2, a splice variant or a fragment thereof.

The invention further encompasses a method of inhibiting an interactionof a DC with a T cell by contacting a composition containing DC and Tcells with an effective amount of an agent that inhibits the interactionof BDCA-2, BDCA-3, or BDCA-4 with the T cell.

The invention further encompasses a method of treating inflammation byadministering to a subject in need thereof an amount of an agent thatinhibits the interaction of BDCA-2, BDCA-3, or BDCA-4 with the T celleffective to reduce inflammation in the subject.

The invention further encompasses a method of suppressing the expressionof BDCA-2 in a cell by expressing a BDCA-2 antisense polynucleotide inthe cell.

The invention further encompasses a transgenic animal containing thepolynucleotide or a complement thereof encoding at least 5 contiguousamino acid residues of BDCA-2, a splice variant or a fragment thereof.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows dot plots from the flow cytometric analysis of peripheralblood mononuclear cells (PBMC) isolated by Ficoll-Paque density gradientcentrifugation. In FIG. 1, expression of BDCA-2, BDCA-3 and CD1c(BDCA-1) on PBMC is shown.

FIG. 1A shows staining of PBMC with FITC-conjugated mAb against BDCA-2(AC144), BDCA-3 (AD5-5E8) and CD1c (AD5-8E7), and PE-conjugated mAbagainst the TCRαβ heterodimer, CD14, CD19 and CD56, respectively. Thenumbers indicate the percentage of cells in the respective quadrant.Propidium iodide fluorescence and light scatter signals were used forgating of live cells.

FIG. 1B shows the scatter profile of (a) PBMC, (b) gated BDCA-2⁺ cells,(c) gated BDCA-3+cells and (d) gated CD1c⁺ cells.

FIG. 2 shows that BDCA-2, BDCA-3, BDCA-4 and CD1c (BDCA-1) are expressedon three distinct blood DC subsets. Blood DC were isolated from PBMC bydepletion of CD3, CD11b and CD16 positive cells followed by enrichmentof CD4 positive cells. The purity of blood DC is demonstrated bylight-scatter properties (upper-left dotplot) and anti-HLA-DR-Cy5 vs.anti-Lin-FITC (anti-TCRαβ, CD14, CD19 and CD56) staining (upper-middledotplot). Note that only few lin⁺ cells are present. Expression ofBDCA-2, BDCA-3, BDCA-4 and CD1c on blood DC is characterized in a seriesof two-color stainings with PE- and FITC-conjugated mAb against CD11c,CD123 and the antigens themselves. Note that BDCA-2, BDCA-3, BDCA-4 andCD1c are exclusively expressed on only one of three distinct blood DCsubsets each. The subsets are defined according to staining of blood DCwith CD123-PE vs. CD11c-FITC (upper-left dotplot): CD11c⁻CD123^(bright)blood DC; CD11c^(bright)CD123^(dim) blood DC; and CD11c^(dim)CD123⁻blood DC.

FIG. 3 depicts expression of BDCA-4 on PBMC. Shown is a two-colorstaining of PBMC with FITC-conjugated MAB against BDCA-2 (AC144) andPE-conjugated mAB against BDCA-4 (AD5-17F6). Note that a few singlepositive (BDCA-2+BDCA-4- and BDCA-2-BDCA-4+) PBMC are detected

FIG. 4 shows the expressino of BDCA-2, BDCA-3 and BDCA-4 on purifiedblood DC after various periods of culture in the presence of IL-3.Purified blood DC were cultured for 0 h, 1 h, 3 h, 6 h, 9 h, 12 h, 18 h,24 h, 36 h, and 48 h in the presence of r1L-3 and then flowcytometrically analyzed for the expression of CD11c, BDCAj-3, BDCA-2 andBDCA-4. (A) Histograms show staining of gated CD11c and CD11c⁺ blood DCwith PE-conjugated anti BDCA-2 mAB (AC144) and anti-BDCA-4 mAB(AD5-17F6) (bold lines), and PE-conjugated isotype-matched control mAB(faint lines), respectively. Dot plots show staining of blood DC withCD11c-PE vs. anti BDCA-3 (AD5-5E8) biotin/streptavidin-APC. (B) Diagramsshow mean fluoroescence intensity (MFI) values for anti-BDCA-2-PE, antiBDCA-4-PE, and anti-BDCA-3 biotin/streptavidin-APC staining of CD11c⁻(▴) and CD11c⁺(▪) DC, respectively. For BDCA-2 and BDCA-4, MFI valueswere calculated by subtracting the values obtained with isotype controlmAb from the values obtained with the AC144 and AD5-17F6, respectively.For BDCA-3, MFI values are calculated by subtracting the values obtainedwithout any staining mAb (autofluorescence) from the values obtainedwith AD5-5E8.

FIG. 5 shows the amino acid sequence of one isoform of BDCA-2 with allsix exons being expressed (SEQ ID NO:2).

FIG. 6 shows that BDCA-1-specific mAb AD5-8E7 blocks binding of the CD1cmAb M241 to MOLT-4 cells. MOLT-4 cells were pre-incubated withsaturating amounts of AD5-8E7 mAb (bold line) or an isotope control mAb(faint line) and then stained with PE-conjugated CD1c mAb (M241).

FIG. 7 shows the expression of BDCA-2, BDCA-3 and BDCA-4 on Mo-DC andCD34⁺ cell-derived DC (CD34-DC). CD14⁺ monocytes and CD34⁺ hematopoieticprogenitor cells were immunomagnetically purified via direct magneticlabeling with CD14 and CD34 mAb-conjugated microbeads, respectively.Purified monocytes were cultured for 7 d in the presence of rGM-CSF andrIL-4, and purified CD34-DC were cultured for 11 d in the presence ofrflt3-ligand, rTGF-β1, rTNF-α, rSCF and rGM-CSF. After the cultureperiod, cells were stained with CD1a-FITC, CD1c-PE (AD5-8E7),anti-BDCA-2-PE (AC114), anti-BDCA-3-PE (AD5-5E8) and anti-BDCA-4-PE(AD5-17F6). Histograms show staining of (A) Mo-DC and (B) CD34-DC (boldlines), respectively. The faint lines show staining with isotype controlmAb. Except for the left-most histogram (CD a staining), gated CD1a⁺cells are shown in (B).

FIG. 8 shows that culturing of anti-BDCA-2 mAb-labeled BDCA-2⁺ cellsresults in rapid mAb internalization. PBMC were labeled at 4° C. withFITC-conjugated anti-BDCA-2 mAb (AC144, IgG1), incubated at 37° C. forthe time periods indicated, and were then stained at 4° C. withPE-conjugated rat anti-mouse IgG1 mAb (X56) and Cy5-conjugated CD123 mAb(AC145, IgG2a). Shown are MFI values of anti-BDCA-2-FITC (▪) and ratanti-mouse IgG1 mAb-PE (▴) staining of gated BDCA-2⁺CD123⁺ cells.

FIG. 9 shows the morphology of immunomagnetically purified CD1c⁺,BDCA-2⁺ and BDCA-3⁺ blood DC. CD1c⁺, BDCA-2⁺ and BDCA-3⁺ cells wereisolated from PBMC by indirect magnetic labeling with PE-conjugatedprimary mAb (AD5-8E7, AC144 and AD5-5E8) and anti-PE mAb-conjugatedmicrobeads followed by enrichment of labeled cells by MACS. The dotplotsshow staining of PBMC with HLA-DR-FITC and the PE-conjugated mAb before(left dotplots) and after (right dotplots) magnetic enrichment of CD1c⁺(upper dotplots) BDCA-2⁺ (middle dotplots) and BDCA-3⁺ (lower dotplots)cells, respectively. The three pictures on the right side show MayGrunwald/Giemsa staining of isolated CD1c⁺ (upper picture), BDCA-2⁺(middle picture) and BDCA-3⁺ cells after cytocentrifugation. Note thatsmall lymphocytes can be seen in the picture of the enriched CD1c⁺cells. These are CD1c⁺ B cells.

FIG. 10 shows up-regulation of MHC class II, CD83 and co-stimulatorymolecules on CD1c⁺, BDCA-2⁺ and BDCA-3⁺ blood DC upon culturing.Purified CD1c⁺ (A), BDCA-2⁺ (C) and BDCA-3⁺ (B) were cultured for 1 dayin medium (CD1c⁺ and BDCA-3⁺ BDC) or for 2 days in medium with rIL-3 andanti-CD40 mAb on CD32-transfected L cells (BDCA-2⁺ DC), respectively.“Immature” Mo-DC (D) were generated by culturing of monocytes for 7 daysin medium in the presence of rGM-CSF and rIL-4. “Mature” Mo-DC (E) weregenerated by culturing of immature Mo-DC for another 3 days in medium inthe presence of TNFα. The histograms show cell staining with CD1a-FITC,CD80-PE, CD83-PE, CD86-PE and HLA-DR-PE, respectively (bold lines). Thefaint lines show cell staining with isotype and fluorochrome-matchedcontrol mAb.

FIG. 11 shows endocytic capacity of freshly isolated CD1c⁺, BDCA-2⁺ andBDCA-3⁺ blood DC in comparison with purified CD3⁺ T cells. IsolatedCD1c⁺ DC (♦), BDCA-2⁺ BDC (▴), BDCA-3⁺ DC (▪) and CD3⁺ T cells (*) wereincubated at 37° C. in medium with 1 mg/ml Lucifer Yellow (LY) for 0,15, 45 and 75 min, washed three times in ice cold PBS/EDTA/BSA and werethen analyzed by flow cytometry. Shown are the MFI values for LYfluorescence after subtracting the MFI values, which are obtained uponincubation at 4° C. in the absence of LY.

FIG. 12 depicts the cDNA sequence of BDCA-2 (SEQ ID NO:1).

FIG. 13 shows intracellular Ca²⁺ mobilization is induced inimmunomagnetically purified BDCA-2⁺BDCA-4⁺ blood DC (A, B) andBDCA-2-transfected U937 cells (D), but not in non-transfected U937 cells(E) via anti-BDCA-2 mAb alone (A) and or anti-BDCA-2 plus crosslinkingsecondary mAb (B, D, E). Ligation of BDCA-4 on immunomagneticallypurified BDCA-2⁺BDCA-4⁺ BDC with anti-BDCA-4 mAb and cross-linkingsecondary mAb does not induce intracellular Ca²⁺ mobilization. Shown isthe Ca²⁺-dependent 405 nm/525 nm ratio of Indo-1-fluorescence (Y-axis)against time (X-axis, a value of 1024 corresponds to 204.80 sec). A isBDCA-2+BDCA-4+ blood DC, anti-BDCA-2 (AC144, IgG1). B is BDCA-2+ BDCA-4+blood DC, anti-BDCA-2 (AC144, IgG1) plus rat anti-mouse IgG1 (X56). C isBDCA-2+ BDCA-4+ blood DC, anti-BDCA-4 (AD5-17F6, IgG1) plus ratanti-mouse IgG (X56). D is BDCA-2 transfected U937 cells, anti-BDCA-2(AC144, IgG1) plus rat anti-mouse IgG1 (X56). E is non-transfected U937cells, anti-BDCA-2 (AC144, IgG1) plus rat anti-mouse IgG1 (X56).

FIG. 14 shows ligation of BDCA-2 but not of BDCA-4 with a specific mAbfollowed by a secondary cross-linking mAb inhibits secretion of type Iinterferon by plasmacytoid BDCA-2⁺BDCA-4⁺ DC from blood or tonsils inresponse to stimulation with influenza virus strain PR8. PlasmacytoidBDCA-2⁺BDCA-4⁺ DC from freshly isolated blood (A) or tonsils (B) werecultured for 24 hours in the presence of IL-3 alone (control); IL-3,anti-BDCA-2 mAb and rat anti-mouse IgG1 mAb (AC144+RamG1); IL-3;anti-BDCA-2 mAb, rat anti-mouse IgG1 mAb, and influenza virus strain PR8(AC144+RamG1+FLU); IL-3 and influenza virus strain PR8 (FLU); IL-3,anti-cytokeratin mAb, rat anti-mouse IgG1 mAb, and influenza virusstrain PR8 (CK3+RamG1+FLU); IL-3, anti-BDCA-4 mAb, rat anti-mouse IgG1mAb, and influenza virus strain PR8 (17F6+RamG1+FLU). Secreted type Iinterferon (U/ml) in the culture supernatants was measured by a bioassaywith reference to a standard type I interferon (U/ml) curve.

FIG. 15 shows presentation of anti-BDCA-2 mAb (AC144, IgG1) to a T cellclone specific for mouse IgG1 by isolated BDCA-2- and BDCA-4-expressingplasmacytoid DC. BDCA-2⁺BDCA-4⁺ plasmacytoid DC present anti-BDCA-2 mAb(AC144, IgG1, ▪) to T cells much more efficiently than anti-ILT-3 mAb(ZM3.8, IgG1, ▴) and anti-cytokeratin mAb (CK3-11D5, IgG1, ).

FIG. 16 shows expression of BDCA-2 and BDCA-4 on tonsillar plasmacytoidCD123⁺ DC.

FIG. 17 shows that neuropilin-1 (GenBank Accession No. 003873) isimmunoprecipitated from cell lysates of neuropilin-1-transfected PEAcells (NP), but not of non-transfected PAE cells (P) with theanti-BDCA-4 mAb AD5-17F6 (anti-NRP-1(ML)). Precipitated proteins wereanalyzed by SDS-PAGE and Western blotting with the BDCA-4-specific mAbAD5-17F6 (ML) or an neuropilin-1-specific mAb from Shay Soker,Children's Hospital, Boston, Mass. (S).

FIG. 18 shows ligation of BDCA-2 but not of BDCA-4 with a specific mAbfollowed by a secondary cross-linking mAb inhibits secretion of INF-α byplasmacytoid BDCA-2⁺BDCA-4⁺ DC from blood or tonsils in response tostimulation with poly I:C. Plasmacytoid BDCA-2+ BDCA-4+ DC from bloodwere cultured with 10 μg/ml of AC144 mAb (2 and 4) or mouse IgG1 mAb(CF6B, anti-TPO, 1 and 3) at 37° C. for 30 min.

FIG. 19 shows an analysis of human multiple tissue cDNA panels fromCLONTECH (lane 1: heart; lane 2: brain; lane 3: placenta; lane 4: lung;lane 5: liver; lane 6: skeletal muscle; lane 7: kidney; lane 8:pancreas; lane 9: spleen; lane 10: thymus; lane 11: testis; lane 12:ovary; lane 13: small intestine; lane 14: lymph node; lane 15: bonemarrow; lane 16: fetal liver; lane 17: tonsil) and an analysis of cDNAsprepared from different populations of blood leukocytes (lane 18: Tcells; lane 19: B cells; lane 20: NK cells; lane 21: monocytes; lane 22:CD11c^(bright)CD123^(low) BDC; lane23: CD11c-CD123^(bright) plasmacytoidDC) for BDCA-2 cDNA. The control is G3PDH.

FIG. 20 shows the splice variants of the BDCA-2 transcript. Splicevariants were analyzed by RT-PCR using the specific primers for BDCA-2used in expression analysis. The amplified fragments were cloned toplasmid vectors and sequenced.

FIG. 21 shows the splice variants of Dectin-2 transcripts.

FIG. 22 shows an alignment of the mRNA sequences of BDCA-2 (SEQ ID NO:1)and mouse Dectin-2 (SEQ ID NO:3) with the precise positions of thededuced introns indicated.

FIG. 23 shows the alignment of the amino acid sequences of human BDCA-2(SEQ ID NO:2), human DCIR (SEQ ID NO:5) and mouse Dectin-2 (SEQ IDNO:4). In FIG. 23, * represents identical conserved residues in all thealigned sequences, : represents conserved substitutions, representssemi-conserved substitutions, shaded areas denote the conservedcarbohydrate recognition domain (CRD), italics show putativetransmembrane domains. The following symbols highlight residues stronglyconserved between C-type lectins in the CRD:

H hydrophobic A Aliphatic C Cysteine G Glycine E glutamic acid Wtryptophan Δ aromatic amino acid + residues involved incalcium-dependent binding of carbohydrates +P++ region determiningcarbohydrate-binding specificity

FIG. 24 shows BDCA-3 immunoprecipitated from cell lysates of P surfacebiotinylated HD-MY-Z cells with the BDCA-3-specific mAb AD5-14H12(IgG1). For control of specificity, the CD19-specific mAb SJ25-C1 (IgG1)was used. Precipitated proteins were analyzed by SDS-PAGE (4-12%) andWestern blotting with streptavidin-peroxidase. Note that theBDCA-3-specific mAb AD5-14H12 specifically immunoprecipitates a cellsurface protein of about 100 kD from HD-MY-Z cells. Thus, BDCA-3 has anapparent molecular weight of 100 kD.

SEQUENCE IDENTIFIERS ARE ASSIGNED AS FOLLOWS

-   -   SEQ ID NO: 1 refers to human BDCA-2 cDNA sequence.    -   SEQ ID NO: 2 refers to mouse BDCA-2 amino acid sequence.    -   SEQ ID NO: 3 refers to mouse Dectin-2 cDNA sequence.    -   SEQ ID NO: 4 refers to mouse Dectin-2 cDNA sequence.    -   SEQ ID NO: 5 refers to human DCIR amino acid sequence.    -   SEQ ID NO: 6 refers to basic unit of a linking peptide (GGGGS).    -   SEQ ID NO: 7 refers to BDCA-2 forward primer (ttgaaagaac        cacaccccga aagt).    -   SEQ ID NO: 8 refers to BDCA-2 reverse primer (tagctttcta        caacggtgga tgcc).    -   SEQ ID NO: 9 refers to BDCA-2 ASN glycosylation domain (NCSV).    -   SEQ ID NO: 10 refers to BDCA-2 ASN glycosylation domain (NSSY).    -   SEQ ID NO: 11 refers to BDCA-2 ASN glycosylation domain (NVTF).    -   SEQ ID NO: 12 refers to Dectin-2 ASN glycosylation domain        (NESL).    -   SEQ ID NO: 13 refers to DCIR ASN glycosylation domain (NESS).    -   SEQ ID NO: 14 refers to BDCA-2 cAMP- and cGMP-dependent protein        kinase phosphorylation site domain (KRLS).    -   SEQ ID NO: 15 refers to DCIR cAMP- and cGMP-dependent protein        kinase phosphorylation site domain (KKTT).    -   SEQ ID NO: 16 refers to BDCA-2 Casein kinase II phosphorylation        site domain (TREE).    -   SEQ ID NO: 17 refers to BDCA-2 Casein kinase II phosphorylation        site domain (SSEE).    -   SEQ ID NO: 18 refers to Dectin Casein kinase II phosphorylation        site domain (STKE).    -   SEQ ID NO: 19 refers to Dectin Casein kinase II phosphorylation        site domain (STSE).    -   SEQ ID NO: 20 refers to Dectin Casein kinase II phosphorylation        site domain (TEAE).    -   SEQ ID NO: 21 refers to Dectin Casein kinase II phosphorylation        site domain (SICE).    -   SEQ ID NO: 22 refers to DCIR Casein kinase II phosphorylation        site domain (TYAE).    -   SEQ ID NO: 23 refers to DCIR Casein kinase II phosphorylation        site domain (TTKE).    -   SEQ ID NO: 24 refers to DCIR Casein kinase II phosphorylation        site domain (TTLE).    -   SEQ ID NO: 25 refers to DCIR Casein kinase II phosphorylation        site domain (SWQD).    -   SEQ ID NO: 26 refers to DCIR Casein kinase II phosphorylation        site domain (SEKD).    -   SEQ ID NO: 27 refers to DCIR Casein kinase II phosphorylation        site domain (TQEE).    -   SEQ ID NO: 28 refers to DCIR Casein kinase II phosphorylation        site domain (SDPE).    -   SEQ ID NO: 29 refers to DCIR Casein kinase II phosphorylation        site domain (SVCE).    -   SEQ ID NO: 30 refers to BDCA Tyrosine kinase phosphorylation        site domain (KLREYQQY).    -   SEQ ID NO: 31 refers to mouse Dectin Tyrosine kinase        phosphorylation site domain (RRLYELHTY).    -   SEQ ID NO: 32 refers to BDCA-2 Amidation site domain (GGRR).    -   SEQ ID NO: 33 refers to mouse Dectin N-myristylation site        (GVCWTL).    -   SEQ ID NO: 34 refers to mouse Dectin N-myristylation site        (GTMVSE).    -   SEQ ID NO: 35 refers to mouse Dectin N-myristylation site        (GCCPNH).    -   SEQ ID NO: 36 refers to DCIR N-myristylation site (GINTAS).    -   SEQ ID NO: 37 refers to consensus immunoreceptor tyrosine-based        inhibitory motif ITIM motif, (I/V)XYXX(L/V).    -   SEQ ID NO: 38 refers to the ITIM motif in DCIR (ITYAEV).

DETAILED DESCRIPTION OF THE INVENTION

The invention relates to methods of enriching for cell populationsenriched in DCs and subsets thereof. Compositions enriched for the DCsand populations of cells obtained therefrom are also provided by theinvention. Methods and compositions for modified cells are alsoprovided. Compositions of modified cells, including genetically modifiedcells are also provided. Methods of use of the cells both modified andnon-modified are provided. Antigen-binding fragments and the antigensrecognized thereby are also provided.

Described herein is a panel of new mAb raised against immunomagneticallypurified CD4⁺ lin⁻ DC that identify three DC antigens: BDCA-2, BDCA-3and BDCA-4. BDCA-2 and BDCA-3 are novel. In the case of BDCA-4, whilenot previously described as a DC-specific antigen, the antigen has beenidentified as neuropilin-1, a receptor for the collapsin/semaphorinfamily that mediates neuronal cell guidance. He et al. (1997) Cell90:739-751.

In non-cultured human blood, expression of BDCA-2 and BDCA-4 is strictlyconfined to plasmacytoid CD123^(bright)CD11c⁻ DC, whereas expression ofBDCA-3 is restricted to a small population of CD123⁻CD11c^(dim) DC. ThisBDCA-3⁺ DC population shares many immunophenotypic features withclassical CD123^(dim)CD11c^(bright) DC, but, unlikeCD123^(dim)CD11c^(dim) DC, BDCA-3⁺ DC lack expression of CD1c (BDCA-1),CD2 and several of the Fc receptors.

The unpurified source of DCs may be any known in the art, such as thebone marrow, fetal, neonate or adult or other hematopoietic cell source,e.g., fetal liver, peripheral blood or umbilical cord blood tonsil,lymph node, nasal membrane, spleen, skin, airway epithelia, lung, livergut, Peyers patches, etc. DCs can also be isolated from cultured cellssuch as DCs derived from progenitor cells. Various techniques can beemployed to separate the cells. For instance, negative selection methodscan remove non-DCs initially. mAbs are particularly useful foridentifying markers associated with particular cell lineages and/orstages of differentiation for both positive and negative selections.

If desired, a large proportion of terminally differentiated cells can beinitially removed using a relatively crude negative separation. Forexample, magnetic bead separations can be used initially to remove largenumbers of irrelevant cells. At least about 80%, usually at least 70% ofthe total cells will be removed prior to isolation of DCs. Preferably,the DC are directly isolated from the cell source by positive selection

Procedures for separation include, but are not limited to, densitygradient centrifugation; rosetting; coupling to particles that modifycell density; magnetic separation with antibody-coated magnetic beads orantibody-coated fero fluids (nonoporticles); affinity chromatography;cytotoxic agents joined to or used in conjunction with a mAb, including,but not limited to, complement and cytotoxins; and panning with antibodyattached to a solid matrix, e.g. plate, elutriation or any otherconvenient technique.

Techniques providing accurate separation and analysis include, but arenot limited to, magnetic bead separation and flow cytometry, which canhave varying degrees of sophistication, e.g., a plurality of colorchannels, low angle and obtuse light scattering detecting channels,impedance channels, etc.

The cells can be selected against dead cells, by employing dyesassociated with dead cells such as propidium iodide (PI). Preferably,the cells are collected in a medium comprising 2% serum, such as fetalcalf serum (FCS) or, human serum albumin (HSA) or any other suitable,preferably sterile, isotonic medium. For physiologic indications, HAS ispreferred. Genetic modification of the cells can be accomplished at anypoint during their maintenance by transducing a substantiallyhomogeneous cell composition with a recombinant DNA construct,transfected with RNA, cell fusion, loading with antigens and variousmethods known in the and/or described herein.

For modification of the cells, a retroviral vector can be employed,however any other suitable vector, delivery system or cellularmodification can be used. These include, e.g., adenovirus,adeno-associated virus, artificial chromosomes, derived from yeast andRNA derived from an antigen source such as a tumor. The geneticmodification, if any, need not be permanent as mature DCs have a limitedlifetime. Genetic approaches are used to express foreign (tumor, viral,parasitic, etc.) antigens or autoantigens in DCs in order to induceimmunity or tolerance. The longevity of the modification can also becontrolled by suicide genes to limit therapy (as with T cells).

Methods of transduction include any known in the art including, withoutlimitation, direct co-culture of the cells with producer cells, e.g., bythe method described by Bregni et al. (1992) Blood 80:1418-1422, orculturing with viral supernatant alone with or without appropriategrowth factors and polycations, e.g., by the method described by Xu etal. (1994) Exp. Hemat. 22:223-230; and Hughes et al. (1992) J. Clin.Invest. 89:1817.

Upon reintroduction of the modified cells expressing or loaded with anantigen so as to present the antigen, into the host, T cells areactivated, anergized or deleted and are specifically directed againstthe antigen. Generally, suitable antigens include those expressed byvirally infected cells, or cancer cells, bacteria, yeast, protozoan,autoantigens (tolerogens) and allergens. More specifically, suitableantigens include, but are not limited to, viral proteins, proteins ofcancer cells, tissue-specific proteins or tolerogenic proteins.“Induction” of T cells can include inactivation of antigen-specific Tcells such as by deletion or anergy. Inactivation is particularly usefulto establish or reestablish tolerance such as in organ transplantationand autoimmune disorders respectively. The modified DCs can beadministered by any method known in the art including, but not limitedto, intravenously, subcutaneously, intranodally and directly to thethymus. Preferably, administration is intravenous (IV).

Often, cell immunotherapy involves removal of bone marrow leukopheresisharvests or other source of cells from a human host, isolating the cellsfrom the source. Meanwhile, the host may be treated to partially,substantially or completely ablate native hematopoietic capability ifhematopoietic stem cell transplantation is to occur. The isolated cellscan be modified during this period of time, so as to provide for cellshaving the desired modification. In the case of complete hematopoieticablation, stem cell augmentation will also be required. The cells ormodified cells can then be restored to the host to provide for the newcapability. The methods of cell removal, host ablation andstem/progenitor cell repopulation are known in the art.

The modified cells can be administered in any physiologically acceptablevehicle, normally intravascularly, intranodal and subcutaneously.Usually, at least 1×10⁵ cells will be administered, preferably 1×10⁶ ormore. The cells can be introduced by injection, catheter, or the like.If desired, factors can also be included, including, but not limited to,interleukins, e.g. IL-2, IL-3, IL-4, IL-12, and flt-Ligand, as well asthe other interleukins, the colony stimulating factors, such as G-, M-and GM-CSF, interferons, e.g. γ-interferon.

The term “polypeptide”, “peptide” and “protein” are used interchangeablyherein to refer to polymers of amino acid residues of any length. Thepolymer can be linear or branched, it can comprise modified amino acidresidues or amino acid analogs, and it can be interrupted by chemicalmoieties other than amino acid residues. The terms also encompass anamino acid polymer that has been modified naturally or by intervention;including, but not limited to, disulfide bond formation, glycosylation,lipidation, acetylation, phosphorylation, or any other manipulation ormodification, such as conjugation with a labeling or bioactivecomponent. Unless stated or implied otherwise, the term antigen-bindingfragment includes any polypeptide monomer or polymer with immunologicspecificity, including the intact antibody, and smaller and largerfunctionally equivalent polypeptides, as described herein.

1. Antigen-Binding Fragments and Compositions Thereof

This invention encompasses antigen-binding fragments that specificallyrecognize DCs. That is, the antigen is found on DCs such thatantigen-binding fragments that recognize the antigen preferentiallyrecognize or bind to DCs or a subset thereof. Or, as with BDCA-4, theantigen may be found on other cell types; but within hematopoieticcells, the antigen is predominately present on DCs.

The invention further encompasses a composition of matter comprising anisolated antigen-binding fragment that binds specifically to at leastone DC antigen. Preferably, the antigen-binding fragment is or isderived from a mAb designated AC144, AD5-13A11, AD5-20E5, AD5-17F6,AD5-4B8, AD5-5E8, AD5-14H12 and AD5-8E7. Table 1 shows the antigen andepitope recognized by each mAb and the isotype of the mAbs specific forDC.

TABLE 1 CD11c^(bright) CD11c^(low) CD11c⁻ CD123^(low) CD123⁻CD123^(bright) Other Antigen Antibody Epitope Isotype DC DC DCleukocytes CD1c AD5-8E7 1A IgG2a + − − Bcell subset BDCA-2 AC144 2A IgG1− − + − BDCA-2 AD5-13A11 2A IgG2a − − + − BDCA-2 AD5-5B8 2A IgG1 − − + −BDCA-3 AD5-5E8 3A IgG1 − + − − BDCA-3 AD5-14H12 3B IgG1 − + − − BDCA-4AD5-17F6 4A IgG1 − − + −

In non-cultured human blood, BDCA-2 and BDCA-4 are expressed by aCD123^(bright)CDC11c⁻ DC population. This DC population is now commonlyreferred to as plasmacytoid DC. Using BDCA-2 or BDCA-4 as a surfacemarker for immunomagnetic isolation and/or flow cytometricidentification of plasmacytoid DC, the results presented herein onfrequency, immunophenotype, morphology, endocytic capacity, andmaturation of these cells, were completely consistent with previousreports, where a large panel of leukocyte antigens was used. Thisclearly illustrates that both antigens are useful markers forplasmacytoid DC in non-cultured human blood. Stainings of tonsillarcells show (FIG. 16) that the T cell zone associated plasmacytoid DC inperipheral lymphoid organs can also be discriminated from other lymphoidtissue-associated DC populations, such as germinal center DC,interdigitating DC and follicular DC based on the expression of BDCA-2and BDCA-4.

Unlike BDCA-2, BDCA-4 is also expressed on several in vitrodifferentiated DC populations: (1) in contrast to BDCA-2, BDCA-4 isexpressed on both Mo-DC and CD34-DC; (2) whereas expression of BDCA-2 iscompletely down-regulated on plasmacytoid DC once they have undergoneIL-3-mediated maturation in culture, expression of BDCA-4 is in factup-regulated on cultured plasmacytoid DC; and (3) in contrast to BDCA-2,BDCA-4 becomes expressed within 12 h by a majority of cultured CD11c⁺DC, whereby it is unclear whether this is only true for the largerCD1c⁺CD11c^(bright) population or also true for the smallerCD1c⁻CD11C^(dim)CD123⁻ population. The finding that no other BDCA-4⁺cells than plasmacytoid DC are present in non-cultured human blood, infact, indicates that no counterparts of the in vitro differentiatedBDCA-4⁺ DC populations mentioned above are present in blood.

Cross-linking of BDCA-2 by means of anti-BDCA-2 mAb induces rapidinternalization of the antigen-Ab complex. In analogy to other endocyticreceptors on DC that are down-regulated upon maturation, like Langerin.Valladeau et al. (2000) Immunity 12:71-81. Therefore, BDCA-2 may be areceptor with antigen-capture function. BDCA-2 is a C-type lectin, israpidly internalized after ligation (FIG. 8), and BDCA-2 ligand(s) areprocessed and presented to T cells (FIG. 15). Thus, like DEC-205, BDCA-2has an antigen uptake and presentation function for ligands to T cells.

Expression of BDCA-3 is restricted to a small population ofCD1c-CD11c^(dim)CD123⁻ DC in non-cultured human blood. With respect tophenotype, morphology, endocytic capacity, and maturation requirements,this DC population is quite similar to theCD1c⁺CD11c^(bright)CD123^(dim) DC population. However, apart from BDCA-3and CD1c expression themselves, the immunophenotypic analysis hasrevealed some striking differences: in contrast to CD1c⁺ BDC, BDCA-3⁺BDC do not express the Fc receptors CD32, CD64 and EcoRI, and they donot express CD2. The lack of Fc receptor expression indicates thatBDCA-3⁺ BDC, unlike CD1c⁺ BDC do not have the capability of Ig-mediatedantigen uptake. Fanger et al. (1996) J. Immunol. 157:541-548; Fanger etal. (1997) J. Immunol. 158:3090-3098; and Maurer et al. (1996) J.Immunol. 157:607-616. As shown herein, BDCA-3 is a 100 kD protein.

There is evidence that CD1c⁺CD11c^(bight) DC, in contrast to CD1c⁻CD11c^(dim) DC, have the capacity to acquire Langerhans cellcharacteristics (expression of Lag antigen, E-cadherin and Langerin, andpresence of Birbeck granules) when cultured with GM-CSF, IL-4 andTGF-β1. If BDCA-3⁺ DC and CD1c⁺ DC represent maturational stages of thesame cell type, this would indicate that, BDCA-3⁺ DC have either alreadylost or not yet acquired the capacity to differentiate into Langerhanscells.

In contradiction to the results presented herein, Ito et al. (1999)reported that CD1c⁺CD11c^(bright) DC, unlike CD1c⁻CD11c^(dim) DC,express CD1a. Two mAb BL-6 and B-B5 were used for staining of CD1a andthat a difference in staining intensity was actually observed when thetwo mAb were compared (staining with B-B5 was probably brighter). Asshown herein, staining of DC was clearly negative using optimal titersof the CD1a mAb BL-6 and HI149, but positive using B-B5. Moreover, B-B5,unlike BL-6 and HI149, stained a high proportion of CD19⁺ B cells inblood. Thus, the staining pattern of B-B was quite reminiscent of a CD1cmAb rather than a CD1a mAb and, in fact, CD1c mAb AD5-8E7 inhibitsbinding of B-B5 to MOLT-4 cells. Therefore, we conclude that B-5recognizes CD1c and that CD1c⁺ DC do not express CD1a.

Staining of cD1c⁺ DC for CD1c, CD2 and CD14 revealed that a minorproportion of DC expresses CD14 to a variable degree and that the levelof CD1c as well as CD2 expression on these cells is inverselyproportional to the level of CD14 expression. This observation is inaccordance with a linear differentiation model, whereCD1c⁺CD2⁺CD11c^(bright)CD14⁻ DC are the progeny of CD14⁺CD1c⁻CD2⁻monocytes rather than the progeny of a common precursor of both celltypes. This concept finds further support by the observation that aconsiderable proportion of CD14⁺ monocytes already express very lowlevels of CD2 and have the capacity to rapidly differentiate into matureDC with typical dendritic morphology and potent T cell stimulatoryfunction when cultured with GM-CSF and IL-4. Crawford et al. (1999) J.Immunol. 163:5920-5928.

The use of CD1c (BDCA-1), BDCA-2, BDCA-3 and BDCA-4 mAb provides aconvenient and efficient way to rapidly detect, enumerate and isolate DCpopulations from PBMC, leukapheresis material, whole blood, tonsil,etc., without apparent functional perturbation. This is a valuable aidfor their further functional and molecular characterization and can beuseful in elucidating their interrelationships. Furthermore, the abilityto easily isolate DC populations to homogeneity greatly facilitatestheir clinical use. The antigen-binding fragments are also useful indetecting, enumerating and/or isolating DCs from tissues, bothnon-hematopoietic tissues (including, without limitation, airwayepithelia, skin, gut, lung, and liver) and hematopoietic tissues(including, without limitation, tonsil, spleen, lymph node and thymus).

Hybridomas secreting the antibodies are also encompassed by theinvention as are other cells expressing antigen-binding fragmentsthereof. Also encompassed by the invention are any antigen-bindingfragments that specifically recognize BDCA-2, or BDCA-3 or BDCA-4. Asseen from Table 1 and the Examples provided herein, multiple types ofmAbs can be produced which specifically recognize these antigens. Asalso seen from the results presented herein, the antigen-bindingfragments need not recognize the same epitope on the same antigen. Allsuch antigen-binding fragments and compositions thereof are encompassedby the invention.

The term “antigen-binding fragment” includes any moiety that bindspreferentially to a DC or a sub-population thereof. Suitable moietiesinclude, without limitation, oligonucleotides known as aptomers thatbind to desired target molecules (Hermann and Pantel (2000) Science289:820-825), carbohydrates, lectins, Ig fragments as Fab, F(ab′)₂,Fab′, scFv (both monomer and polymeric forms) and isolated H and Lchains. An antigen-binding fragment retains specificity of the intactIg, although avidity and/or affinity can be altered.

Certain compounds, compositions and methods described herein relategenerally to antibodies and derivatives thereof which having providedthe antigenic determinants herein, can be generated routinely bystandard immunochemical techniques. These include, but are not limitedto, antigen-binding fragments coupled to another compound, e.g. bychemical conjugation, or associated with by mixing with an excipient oran adjuvant. Specific conjugation partners and methods of making themare described herein and known in the art.

Antigen-binding fragments (also encompassing “derivatives” thereof) aretypically generated by genetic engineering, although they can beobtained alternatively by other methods and combinations of methods.This classification includes, but is not limited to, engineered peptidefragments and fusion peptides. Preferred compounds include polypeptidefragments containing the anti-DC CDRs, antibody fusion proteinscontaining cytokine effector components, antibody fusion proteinscontaining adjuvants or drugs, antibody fusion proteins containing tumorcell-derived antigens, viral antigens, bacterial antigens, parasiteantigens, yeast antigens, autoantigens or antigenic peptides (T cellepitopes) derived therefrom, and, single chain V region proteins.Antigen-binding fragments are considered to be of human origin if theyare isolated from a human source, and used directly or cloned andexpressed in other cell types and derivatives thereof or whole humanchromosomes or portions thereof (such as mice with human chromosomesencoding V_(H), D_(H), J_(H), V_(L), JL_(L), C_(H), C_(L) genesegments).

A “fusion polypeptide” is a polypeptide comprising contiguous peptideregions in a different position than would be found in nature. Theregions can normally exist in separate proteins and are brought togetherin the fusion polypeptide; they can normally exist in the same proteinbut are placed in a new arrangement in the fusion polypeptide; or theycan be synthetically arranged. For instance, the invention encompassesrecombinant proteins (and the polynucleotides encoding the proteins orcomplementary thereto) that are comprised of a functional portion of anantigen-binding fragment and another peptide such as a toxin. Methods ofmaking these fusion proteins are known in the art and are described forinstance in WO93/07286.

A “functionally equivalent fragment” of a polypeptide varies from thenative sequence by any combination of additions, deletions, orsubstitutions while preserving at least one functional property of thefragment relevant to the context in which it is being used.

The antigen-binding fragments are useful in palliating the clinicalconditions related to immunologic disorders. The invention furthercomprises polypeptide derivatives of the antigen-binding fragments andmethods for using these compositions in diagnosis, treatment, andmanufacture of novel reagents.

The invention also encompasses antigen-binding fragments conjugated to achemically functional moiety. Typically, the moiety is a label capableof producing a detectable signal. These conjugated antigen-bindingfragments are useful, for example, in detection systems such asquantitation of DCs in various tissues, in various diseases, after stemcell transplantation, and after immunoablative therapy like chemotherapyand radiation, and imaging of DCs for instance in following chemotherapyor autoimmune therapy. Such labels are known in the art and include, butare not limited to, radioisotopes, enzymes, fluorescent compounds,chemiluminescent compounds, bioluminescent compounds, substratecofactors and inhibitors and magnetic particles. For examples of patentsteaching the use of such labels, see, for instance U.S. Pat. Nos.3,817,837; 3,850,752; 3,939,350; 3,996,345; 4,277,437; 4,275,149; and4,366,241. The moieties can be covalently linked, recombinantly linked,or conjugated (covalently or non-covalently) through a secondaryreagent, such as a second antibody, protein A, or a biotin-avidincomplex.

Other functional moieties include, without limitation, signal peptides,agents that enhance immunologic reactivity, agents that facilitatecoupling to a solid support, vaccine carriers, bioresponse modifiers,paramagnetic labels and drugs. Signal peptides include prokaryotic andeukaryotic forms. Agents that enhance immunologic reactivity include,but are not limited to, bacterial superantigens and adjuvants. Agentsthat facilitate coupling to a solid support include, but are not limitedto, biotin, avidin or derivatives thereof. Immunogen carriers include,but are not limited to, any physiologically acceptable buffer.Bioresponse modifiers include, but are not limited to, cytokines,particularly tumor necrosis factor (TNF), IL-2, interleukin-4 (IL-4),GM-CSF; IL-10, IL-12, TGF-β and certain interferons, and chemokines(MIP-3β, SDF-1, Lymphotactin, DC-CK1, Eotaxins, IP-10, TARC, Rantes,MIP-1x, MIP-1B, SLC, 1-TAC, MIG, MDC, MCP-1, TCA-3, MCP-2, -3, -1. Seealso, U.S. Pat. No. 5,750,119; and WO patent publications: 96/10411;98/34641; 98/23735; 98/34642; 97/10000; 97/10001; and 97/06821. Such,chemokines may be useful to attract other cells such as T cells.

A “signal peptide” or “leader sequence” is a short amino acid sequencethat directs a newly synthesized protein through a cellular membrane,usually the endoplasmic reticulum (ER) in eukaryotic cells, and eitherthe inner membrane or both inner and outer membranes of bacteria. Signalpeptides are typically at the N-terminus of a polypeptide and areremoved enzymatically between biosynthesis and secretion of thepolypeptide from the cell or through the membrane of the ER. Thus, thesignal peptide is not present in the secreted protein but is presentonly during protein production.

Immunotoxins, including single chain conjugates, can be produced byrecombinant means. Production of various immunotoxins is well known inthe art, and methods can be found, for example, in “MonoclonalAntibody-toxin Conjugates: Aiming the Magic Bullet,” Thorpe et al.(1982) Monoclonal Antibodies in Clinical Medicine, Academic Press, pp.168-190; Vitatta (1987) Science 238:1098-1104; and Winter and Milstein(1991) Nature 349:293-299. Suitable toxins include, but are not limitedto, ricin, radionuclides, pokeweed antiviral protein, Pseudomonasexotoxin A, diphtheria toxin, ricin A chain, fungal toxins such asfungal ribosome inactivating proteins such as gelonin, restrictocin andphospholipase enzymes. See, generally, “Chimeric Toxins,” Olsnes andPihl, Pharmac. Ther. 15:355-381 (1981); and “Monoclonal Antibodies forCancer Detection and Therapy,” eds. Baldwin and Byers, pp. 159-179,224-266, Academic Press (1985).

The chemically functional moieties can be made recombinantly forinstance by creating a fusion gene encoding the antigen-binding fragmentand functional regions from other genes (e.g. enzymes). In the case ofgene fusions, the two components are present within the same gene.Alternatively, antigen-binding fragments can be chemically bonded to themoiety by any of a variety of well known chemical procedures. Forexample, when the moiety is a protein, the linkage can be by way ofhomo- or hetero-bifunctional cross linkers, e.g., SPDP, SMCC,carbodiimide glutaraldehyde, or the like. The moieties can be covalentlylinked, or conjugated, through a secondary reagent, including, but notlimited to, a second antibody, protein A, or a biotin-avidin complex.Paramagnetic moieties and the conjugation thereof to antibodies arewell-known in the art. See, e.g., Miltenyi et al. (1990) Cytometry11:231-238.

Here, we overcame problems described in the art (O'Doherty et al.(1993); and Yamaguchi et al. (1995)) with a recently describedcontralateral footpad immunization procedure. Yin et al. (1997) Blood90:5002-5012. This system utilizes naive antigen-specific T and B cellswhich continuously recirculate among peripheral lymphoid organs as longas they do not encounter antigen, but become immediately retained withina peripheral lymphoid organ for several days, if not weeks, once theyare activated by antigen. Picker et al. (1992) Annu. Rev. Immunol.10:561-591; Butcher et al. (1996) Science 272:60-66; Bradley et al.(1996) Curr. Opin. Immunol. 8:312:320; Watson et al. (1998) Cell. Adhes.Commun. 6:105-110; Kearney et al. (1994) Immunity 1:327-339; Jacob etal. (1992) J. Exp. Med. 176:679-687; Ridderstaad et al. (1998) J.Immunol. 160:4688-4695; and Tarlinton (1998) Curr. Opin. Immunol.10:245-251. In the examples provided herein, the left footpads of micewere injected on days −3, 0, 4, 7, 11, and 14 with Bristol-8 Blymphoblastoma cells, while the right footpads were injected with DC ondays 0, 4, 7, 11, and 14. Naïve B an T cells with specificity for sharedantigens, e.g. HLA class II molecules, should become activated byBristol-8 cells between d −3 and 0 in the left popliteal lymph node andthereupon be retained there, while all lymphocytes with specificity forantigens unique to DC should remain available for activation after d 0in the right popliteal lymph node.

This immunization technique was combined with a powerful procedure forrapid isolation of large numbers of DC and permitted production a panelof mAb that recognize three novel DC antigens: BDCA-2, BDCA-3 andBDCA-4. The use of antigens in producing additional DC-specificantibodies allows more traditional methods of antibody production to beused with a greater chance of success.

Methods of antibody production and isolation are well known in the art.See, for example, Harlow and Lane (1988) Antibodies: A LaboratoryManual, Cold Spring Harbor Laboratory, New York. General antibodypurification methods include, but are not limited to, salt precipitation(for example, with ammonium sulfate); ion exchange chromatography (forexample, on a cationic or anionic exchange column run at neutral pH andeluted with step gradients of increasing ionic strength); gel filtrationchromatography (including gel filtration HPLC); and chromatography onaffinity resins such as protein A, protein G, hydroxyapatite, oranti-Ig. Antigen-binding fragments can also be purified on affinitycolumns comprising DCs or an antigenic portion thereof. Preferablyfragments are purified using Protein-A-CL-Sepharose™4B chromatographyfollowed by chromatography on a DEAE-Sepharose™4B ion exchange column.

The invention also encompasses hybrid antibodies, in which one pair of Hand L chains is obtained from a first antibody, while the other pair ofH and L chains is obtained from a different second antibody. Forpurposes of this invention, one pair of L and H chains is from anti-DCantibody. In one example, each L-H chain pair binds different epitopesof a DC-specific antigen. Such hybrids can also be formed usinghumanized H or L chains. The invention also encompasses other bispecificantibodies such as those containing two separate antibodies covalentlylinked through their constant regions.

Other antigen-binding fragments encompassed by this invention areantibodies in which the H or L chain has been modified to provideadditional properties. For instance, a change in amino acid sequence canresult in reduced immunogenicity of the resultant polypeptide. Thechanges range from changing one or more amino acid residues to thecomplete redesign of a region such as a C region domain. Typical changesinclude, but are not limited to, those related to complement fixation,interaction with membrane receptors, and other effector functions. Arecombinant antibody can also be designed to aid the specific deliveryof a substance (such as a cytokine) to a cell. Also encompassed by theinvention are peptides in which various Ig domains have been placed inan order other than that which occurs in nature.

The size of the antigen-binding fragments can be only the minimum sizerequired to provide a desired function. It can optionally compriseadditional amino acid sequence, either native to the antigen-bindingfragment, or from a heterologous source, as desired. Anti-DCantigen-binding fragments can contain only 5 consecutive amino acidresidues from an antibody V region sequence. Polypeptides comprising 7amino acid residues, more preferably about 10 amino acid residues, morepreferably about 15 amino acid residues, more preferably about 25 aminoacid residues, more preferably about 50 amino acid residues, morepreferably about 75 amino acid residues from the antibody L or H chain Vregion are also included. Even more preferred are polypeptides,comprising the entire antibody L or H chain V region.

Substitutions can range from changing or modifying one or more aminoacid residue to complete redesign of a region, such as the V region.Amino acid residue substitutions, if present, are preferablyconservative substitutions that do not deleteriously affect folding orfunctional properties of the peptide. Groups of functionally relatedamino acid residues within which conservative substitutions can be madeare glycine/alanine; valine/isoleucine/leucine; asparagine/glutamine;aspartic acid/glutamic acid; serine/threonine/methionine;lysine/arginine; and phenylalanine/tyrosine/tryptophan. Antigen-bindingfragments can be glycosylated or unglycosylated, can be modifiedpost-translationally (e.g., acetylation, and phosphorylation) or can bemodified synthetically (e.g., the attachment of a labeling group).

Polypeptide derivatives comprising both an L chain and an H chain can beformed as separate L and H chains and then assembled, or assembled insitu by an expression system for both chains. Such expression systemscan be created by transfecting with a plasmid comprising separatetranscribable regions for the L and H chain, or by co-transfecting thesame cell with plasmids for each chain. In a third method, a suitableplasmid with an H chain encoding region is transfected into an H chainloss mutant.

H chain loss mutants can be obtained by treating anti-DC antibodyproducing cells with fluorescein-labeled rabbit anti-mouse IgG (H chainspecific, DAKO Corporation, Carpinteria, Calif.) according to thesupplier's instruction. The stained and unstained cell populations areanalyzed by flow cytometry. Unstained cells are collected in asterilized tube and placed in 96-well plates at 1 cell/well by limitingdilution. Culture supernatants are then assayed by ELISA using goatanti-mouse IgG (H chain specific) and goat anti-mouse kappa. Cloneshaving a kappa-positive, IgG-negative phenotype are subcloned at least 3times to obtain stable anti-DC^((−H)) mutants. mRNA from putative Hchain loss mutants can be isolated and the sequence of the L chain Vregion cDNA determined. Reverse PCR of the mRNA for the V_(H) isperformed with 2 sets of 5′- and 3′-primers, and used for cloning ofanti-DC^((−H)) cDNA. An H chain loss mutant yields no detectable DNAband with these primers. Transfection of the cells proceeds with asuitable H chain plasmid.

Another antigen-binding fragment derivative encompassed by thisinvention is an antibody in which the constant region of the H or Lchain has been modified/to provide additional properties. For instance,a change in amino acid sequence can result in altered immunogenicity ofthe resultant polypeptide. The changes range from one or more amino acidresidues to the complete redesign of constant region domain. Changescontemplated affect complement fixation, interaction with membranereceptors, and other effector functions. A recombinant antibody can alsobe designed to aid the specific delivery of a substance (such as alymphokine or an antigen or an antigenic peptide derived from a tumor,virus, parasite or bacteria, or tolerogen (autoantigen)) to a cell. Alsoencompassed by the invention are proteins in which various Ig domainshave been placed in an order other than that which occurs in nature.

The invention also encompasses single chain V region fragments (“scFv”)of anti-DC antibodies. Single chain V region fragments are made bylinking L and/or H chain V regions by using a short linking peptide.Bird et al. (1988) Science 242:423-426. Any peptide having sufficientflexibility and length can be used as a linker in a scFv. Usually thelinker is selected to have little to no immunogenicity. An example of alinking peptide is (GGGGS)₃ (SEQ ID NO:6), which bridges approximately3.5 nm between the carboxy terminus of one V region and the aminoterminus of another V region. Other linker sequences can also be used,and can provide additional functions, such as a for attaching to a drugor solid support or specific delivery of a substance (such as alymphokine or an antigen or an antigenic peptide derived from a tumor,virus, parasite or bacteria, or tolerogen (autoantigen)) to a cell.

All or any portion of the H or L chain can be used in any combination.Typically, the entire V regions are included in the scFv. For instance,the L chain V region can be linked to the H chain V region.Alternatively, a portion of the L chain V region can be linked to the Hchain V region, or portion thereof. Also contemplated are scFvs in whichthe H chain V region is from an antibody described herein, and the Lchain V region is from another Ig. A biphasic, scFv can be made in whichone component is an antigen-binding fragment and another component is adifferent polypeptide, such as a T cell epitope.

The scFvs can be assembled in any order, for example,V_(H)-(linker)-V_(L) or V_(L)-(linker)-V_(H). There can be a differencein the level of expression of these two configurations in particularexpression systems, in which case one of these forms can be preferred.Tandem scFvs can also be made, such as (X)-(linker)-(X)-(linker)-(X), inwhich X are scFvs, or combinations thereof with other polypeptides. Inanother embodiment, single chain antibody polypeptides have no linkerpolypeptide, or just a short, inflexible linker. Possible configurationsare V_(L)—V_(H) and V_(H)—V_(L). The linkage is too short to permitinteraction between V_(L) and V_(H) within the chain, and the chainsform homodimers with a V_(L)/V_(H) antigen-binding site at each end.Such molecules are referred to as “diabodies.”

ScFvs can be produced recombinantly or synthetically. For syntheticproduction of scFv, an automated synthesizer can be used. Forrecombinant production of scFv, a suitable plasmid-containingpolynucleotide that encodes the scFv can be introduced into a suitablehost cell, either eukaryotic, such as yeast, plant, insect or mammaliancells, or prokaryotic, such as Escherichia coli, and the expressedprotein can be isolated using standard protein purification techniques.ScFvs can also be obtained from a phage display library.

A particularly useful system for the production of scFvs is plasmidpET-22b(+) (Novagen, Madison, Wis.). Escherichia coli pET-22b(+)contains a nickel ion binding domain consisting of 6 sequentialhistidine residues, which allows the expressed protein to be purified ona suitable affinity resin. Another example of a suitable vector ispcDNA3 (Invitrogen, San Diego, Calif.).

Conditions of gene expression preferably ensure that the scFv assumesoptimal tertiary structure. Depending on the plasmid used (especiallypromoter activity), and the host cell, it can be necessary to modulateproduction rate. For instance, use of a weaker promoter, or expressionat lower temperatures, can be necessary to optimize production ofproperly folded scFv in prokaryotic systems; or, it can be used toexpress scFv in eukaryotic cells.

The invention also encompasses polymeric forms of antigen-bindingfragments, containing a plurality of DC-specific antigen-bindingfragments. One embodiment is a linear polymer of antigen-bindingfragments, optionally conjugated to carrier. These linear polymers cancomprise multiple copies of a single antigen-binding fragmentpolypeptide, or combinations of different polypeptides, and can havetandem polypeptides, or polypeptides separated by other amino acidsequences.

Another embodiment is multiple antigen peptides (MAPs). MAPs have asmall immunologically inert core having radially branching-lysinedendrites, onto which a number of antigen-binding fragment polypeptidesare covalently attached. See for instance, Posnett et al. (1988) J.Biol. Chem. 263:1719-1725; and Tam (1989) Met. Enz. 168:7-15. The resultis a large macromolecule having a high molar ratio of antigen-bindingfragment polypeptides to core. MAPs are efficient immunogens and usefulantigens for immunoassays. The core for creating MAPs can be made bystandard peptide synthesis techniques, or obtained commercially (QualityControlled Biochemicals, Inc., Hopkinton, Mass.). A typical core matrixis made up of three levels of lysine and eight amino acid residues.

The invention further includes anti-idiotypic antigen-binding fragmentsto the DC-specific antigen-binding fragments of the invention. Suchanti-idiotypes can be made by any method known in the art.

Cancer patients are often immunosuppressed and tolerant to sometumor-associated antigens (TAA). Triggering an active immune response tosuch TAA represents an important challenge in cancer therapy.Immunization with a given antigen generates an immune response includinga CTL response, preferably a strong CTL response. The production ofantibodies against the antigen can be helpful if the tumor cells arekilled by ADCC (antibody-dependent cellular cytotoxicity). The inventionencompasses the use of DCs identified and isolated by use of theantigen-binding fragments of the invention in inducing specific immuneresponses by methods known in the art. The immune responses can bespecific to any antigen including, without limitation, those associatedwith cancer, infectious viruses, infectious bacteria, infectiousparasites, infectious yeast, and autoimmune diseases (the inducetolerance). The ability to isolate subpopulations that are uniquelysuited to inducing such a response results in preparations of DCs thatare more effective than mixtures of subpopulations. Hybrid cells (e.g.DC/tumor cell) could also be used as cancer-specific therapy. Modifiedcells (including, without limitation, activated, in vitro matured,modulated with respect to their T helper cell polarizing capacity (Th1 vTh2 v Th3/Th-R), and modulated with respect to their T cell stimulatingor anergizing or deleting capacity) are likewise encompassed by theinvention and include, but are not limited to, genetically modified ortransfected cells and cells that have been incubated with peptides orproteins suitable for antigen presentation or for internalization.Subpopulations include, without limitation, a particular differentiationstage within one lineage and a separate lineage of differention.

The invention further provides DCs, subpopulations thereof and mixturesthereof. The cells are selected using the antigen-binding fragmentsprovided herein by any separation method known in the art. Compositionscomprising the isolated cells are also encompassed by the invention.These include pharmaceutical and therapeutic compositions and any othercomposition containing the isolated cells. The DCs subpopulationsisolated by the methods described herein are preferably substantiallyhomogeneous. That is, cells isolated by a BDCA-specific antigen-bindingfragments are preferably more than about 80% BDCA⁺, more preferably morethan about 90% BDCA⁺ and most preferably more than about 95% BDCA⁺. Ofcourse, subsequent combinations of the cells with other DCs, or otherhematopoietic cells can decrease the percentage of BDCA⁺ cells, suchcombinations are also encompassed by the invention.

Likewise, the DCs obtained by the methods described herein are suitablefor use in any method of treatment known in the art include referenceshere. DCs altered to achieve these methods are also encompassed by theinvention. These methods include, but are not limited to:

a) therapy with isolated DCs to induce specific T cell tolerance(killing or anergy instead of stimulation) in autoimmune diseases,allergies, graft versus host disease (GvHD), allograft rejection. Forinstance, DCs specific for such T cells can be modified to containlysis, inactivating or death-inducing moieties so as to specificallytarget the T cells involved in the unwanted immune response for instanceby antigen labeling or genetic modification such as by CD95Ltransfection. DC specificity for T cells is primarily caused bypresentation of the appropriate T cell epitopes (peptides) via MHC I andIL The particular subsets of DCs with tolerance-inducing functions canbe administered directly to the patient. Peripheral tolerance can bemediated by DCs modified to induce deletion (killing), anergy andsuppression/regulation of T cells;

b) immunomodulation therapy with isolated DCs to induce particularcytokine expression profiles in specific T cells. This is particularlyuseful to influence production of Th1 (cytokines for specificinflammatory immune responses), Th2 (cytokines for specific humoralimmune responses) or Th3 (cytokines for specific immunosuppression)cytokines. In the case of allergies and asthma for instance, inductionof a Th1 response may reduce or eliminate the symptom-producing Th2response;

c) therapy with DCs presenting antigens including, but not limited to,tumor antigens, viral antigens and cellular antigens;

d) therapy with DCs (with or without presenting antigens) and variouscofactors including, but not limited to cytokines, costimulatorymolecules and effector molecules in amounts and under conditionssufficient to modulate the immune response; and

e) stimulating T cells in vitro to obtain antigen-specific T cells.

The antigen-binding fragments described herein are also suitable for anumber of methods of treatment. These include, but are not limited to:

a) antibodies mimicking the ligand- or ligand-mediated immunotherapy forinstance of DCs involved in autoimmunity or in vivo targeting ofantigens or nucleic acids (viruses, plasmid DNA, RNA etc) to DCs foroptimal and selective uptake/transfection. BDCA-2 may be particularlyuseful in this context as it appears to be a molecule with antigenuptake and processing function; and

b) immunomonitoring: e.g. enumeration and characterization of BDCA-2⁺,BDCA-3⁺ and BDCA-4⁺ DCs in various diseases and upon mobilization e.g.with a proliferation inducing ligand, e.g. flt3-Ligand or G-CSF.

Any carrier not harmful to the host can be used for the DCs. Suitablecarriers are typically large, slowly metabolized macromolecules such asproteins; polysaccharides (such as latex functionalized Sepharose,agarose, cellulose, cellulose beads and the like); polymeric amino acidresidues (such as polyglutamic acid, polylysine, and the like); aminoacid copolymers; and inactive virus particles or attenuated bacteria,such as Salmonella.

2. Methods of Obtaining Additional Dc-Specific Antigen-Binding Fragments

The invention encompasses methods of obtaining DC-specificantigen-binding fragments.

Methods of generating new DC-specific antigen-binding fragments, asdetailed below, include, but are not limited to: 1) employing phagedisplay techniques by which cDNA encoding antibody repertoires arepreferably amplified from lymphocyte or spleen RNA using PCR andoligonucleotide primers specific for species-specific V regions; 2)immunizing mammals with the antigen and generating polyclonal or mAbs;and 3) employing phage display to make antibodies without priorimmunization by displaying on phage, very large and diverse V generepertoires. See, generally Hoogenboom et al. (1998) Immunotechnol.4:1-20. Preferably, for therapeutic purposes, if non-human antigenbinding fragments are to be used, these can be humanized by any methodknown in the art.

The method described by Medez et al. (1997) Nature Genetics 18:410 canbe used. Briefly, purified antigen, is used to immunize transgenic micelacking the native murine antibody repertoire and instead having most ofthe human antibody V-genes in the germ line configuration. Humanantibodies are subsequently produced by the murine B cells. The antibodygenes are recovered from the B cells by PCR library selection or classichybridoma technology.

Alternatively, antibodies can be obtained from mice (such as BALB/c)after injection with purified DC-specific antigen. mAbs are generatedusing standard hybridoma technology. Maiti et al. (1997) Biotechnol.Int. 1:85-93 (human hybridomas); and Kohler and Milstein (1975) Nature256:495-497 (mouse hybridomas). Murine antibodies can be subsequentlyhumanized for instance by the methods described by Rosok et al. (1996)J. Biol. Chem. 271:22611-22618; Baca et al. (1997) J. Biol. Chem.272:10678-10684; Rader et al. Proc. Natl. Acad. Sci. USA 95:8910-8915;and Winter and Milstein (1991) Nature 349:293-299.

A phage display approach can also be used to rapidly generate humanantibodies against DCs. This approach can employ the method described byHenderikx et al. (1998) Cancer Res. 58:4324-32. Antibody fragmentsdisplayed on phage are selected from a large naïve phageantibody/fragment library containing different single chain antibodiesby separating those that bind to immobilized antigen or DCs. Humanantibody fragments are selected from naïve repertoires constructedeither from germline V-domains or synthesized with many mutations(mutations are targeted either by homologous gene re-assortments orerror prone PCR) in both the framework and CDR regions. Antigen-bindingfragments specifically reactive with DCs can be identified by screeningagainst tumor and normal cells as described herein in order to identifyDC-specific antigen-binding fragments.

The invention also encompasses methods of identifying antigen-bindingfragments specific for a DCs by generating a suitable phage displaylibrary; isolating DC-specific antigens; screening the phage displaylibrary with the antigens according to standard immunochemicaltechniques to obtain phage that display an antigen-binding fragment thatbinds specifically to DCs; or screening the phage display libraryobtained for DC specific antigen-binding fragments, by screening againstDCs and other, related cells such as APCs and selecting the phage thatbind preferentially to DCs. Methods of generating antigen-bindingfragments by phage display are well known in the art. Hoogenboom et al.(1998).

Lymphocyte (PBL) or spleen RNA is typically used to make antibodyfragment repertoires. Mutagenesis using homologous reassortment or errorprone PCR increases the diversity. Any method known in the art can beused.

Repertoires of antibody genes can be amplified from immunized mice orhumans using PCR and scFv or Fab antibody fragments obtained thereby canbe cloned and expressed on the surface of bacteriophage. The antibodygene repertoires are amplified from lymphocyte or spleen RNA using PCRand oligonucleotide primers specific for host animal-specific V regions.Phage display can also be used to make antibodies without priorimmunization by displaying very large and diverse V gene repertoires onphage. The natural V gene repertoires present in PBL are isolated by PCRamplification and the VH and VL regions are spliced together at randomusing PCR. Mutations can be targeted to the V-domain genes by homologousgene reassortments or error-prone PCR. Zhao et al. (1998) Nat.Biotechnol. 15:258; and Hoogenboom et al. (1998). Totally synthetichuman libraries can also be created and used to screen for DC-specificantibody fragments. Regardless of the method used to operate the phagedisplay library, each resulting phage has a functional antibody fragmentdisplayed on its surface and contains the gene encoding the antibodyfragment in the phage genome. See, e.g. WO97/02342.

Affinity chromatography in which binding antibodies can be subtractedfrom non-binding antibodies has been established for some time. Nissimet al. (1994) EMBO J. 13:692-698; and Vaughan et al. (1996) Nat.Biotechnol. 14:309-314. Critical parameters affecting success are thenumber and affinity of antibody fragments generated against a particularantigen. Until recently, the production of large, diverse librariesremained somewhat difficult. Historically, scFv repertoires have beenassembled directly from VH and VL RT-PCR products. RNA availability andthe efficiency of RT-PCR were limiting factors of the number of V genesavailable. Also, assembly required ligating three fragments, namely VHand VL and the linker regions. Marks et al. (1991) J. Mol. Biol.222:581-597.

An improved library construction method uses cloned VH and VL generepertoires in separate plasmid vectors to provide a stable andlimitless supply of material for scFv assembly. Sheets et al. (1998)Proc. Natl. Acad. Sci. USA 95:6175-6162. Also, the efficiency isincreased by having DNA encoding the linker region at the 5′ end of theVL library. Therefore there are only two fragments to be ligated insteadof three.

Anti-DC-antigen-binding fragments can also be derived or manipulated.For example, the immunogenic activity of the V regions of the L and Hchains can be screened by preparing a series of short polypeptides thattogether span the entire V region amino acid sequence. Using a series ofpolypeptides of 20 or 50 amino acid residues in length, each V regioncan be surveyed for useful functional properties. It is also possible tocarry out a computer analysis of a protein sequence to identifypotentially immunogenic polypeptides. Such peptides can then besynthesized and tested.

The invention further encompasses various adaptations of antigen-bindingfragments described herein combined in various fashions to yield otheranti-DC antigen-binding fragments with desirable properties. Forinstance, antigen-binding fragments with modified residues can becomprised in MAPs. In another example, an scFv is fused to a cytokine,such as IL-2. All such combinations are encompassed by this invention.

The antigen-binding fragments can be made by any suitable procedure,including proteolysis, recombinant methods or chemical syntheses. Thesemethods are known in the art and need not be described in detail.Examples of proteolytic enzymes include, but are not limited to,trypsin, chymotrypsin, pepsin, papain, V8 protease, subtilisin, plasmin,and thrombin. Intact antigen-binding fragments can be incubated with oneor more proteases simultaneously or sequentially. Alternatively, or inaddition, intact antibody can be treated with disulfide reducing agents.Peptides can then be separated from each other by techniques known inthe art, including but not limited to, gel filtration chromatography,gel electrophoresis, and reverse-phase HPLC.

Anti-DC antigen-binding fragments can also be made by expression from apolynucleotide encoding the peptide, in a suitable expression system byany method known in the art. Typically, polynucleotides encoding asuitable polypeptide are ligated into an expression vector under controlof a suitable promoter and used to genetically alter the intended hostcell. Both eukaryotic and prokaryotic host systems can be used. Thepolypeptide is then isolated from lysed cells or from the culture mediumand purified to the extent needed for its intended use. Examples ofprokaryotic host cells appropriate for use with this invention includeE. coli, Bacillus subtilis and any other suitable host cell. Examples ofeukaryotic host cells include, but are not limited to yeast, avian,insect, plant, and animal cells such as COS7, HeLa, and CHO cells.

Optionally, matrix-coated channels or beads and cell co-cultures can beincluded to enhance growth of antigen-binding fragment producing cells.For the production of large amounts of mAbs, it is generally moreconvenient to obtain ascitic fluid. The method of raising ascitesgenerally comprises injecting hybridoma cells into an immunologicallynaïve, histocompatible or immunotolerant mammal, especially a mouse. Themammal can be primed for ascites production by prior administration of asuitable composition; e.g., Pristane. The ascitic fluid is removed fromthe animal and processed to isolate antibodies.

Alternatively, antigen-binding fragments can be chemically synthesizedusing amino acid sequence data and other information provided in thisdisclosure, in conjunction with standard methods of protein synthesis. Asuitable method is the solid phase Merrifield technique. Automatedpeptide synthesizers are commercially available, such as thosemanufactured by Applied Biosystems, Inc. (Foster City, Calif.).

Another method of obtaining anti-DC antigen-binding fragments is toimmunize suitable host animals with BDCA-2, BDCA-3 and/or BDCA-4 andfollow standard methods for polyclonal or mAb production and isolation.mAbs thus produced can be “humanized” by methods known in the art. Theinvention thus encompasses humanized mAbs.

In “humanized” antibodies at least part of the sequence has been alteredfrom its initial form to render it more like human Igs. In one version,the H chain and L chain C regions are replaced with human sequence. Thisis a fusion polypeptide comprising an anti-DC V region and aheterologous Ig (C) region. In another version, the CDR regions compriseanti-DC amino acid sequences, while the V framework regions have alsobeen converted human sequences. See, for example, EP 0329400. In a thirdversion, V regions are humanized by designing consensus sequences ofhuman and mouse V regions, and converting residues outside the CDRs thatare different between the consensus sequences.

In making humanized antibodies, the choice of framework residues can aidin retaining high binding affinity. In principle, a framework sequencefrom any human antibody can serve as the template for CDR grafting;however, it has been demonstrated that straight CDR replacement intosuch a framework can lead to significant loss of antigen bindingaffinity. Glaser et al. (1992) J. Immunol. 149:2606; Tempest et al.(1992) Biotechnol. 9:266; and Shalaby et al. (1992) J. Exp. Med. 17:217.The more homologous a human antibody is to the original murine antibody,the less likely that the human framework will introduce distortions intothe murine CDRs that could reduce affinity. Based on a sequence homologysearch against an antibody sequence database, the human antibody IC4provides good framework homology to muM4TS.22, although other highlyhomologous human antibodies are suitable as well, especially κ L chainsfrom human subgroup I or H chains from human subgroup III. Kabat et al.(1987). Various computer programs such as ENCAD predict the idealsequence for the V region. Levitt et al. (1983) J. Mol. Biol. 168:595.The invention thus encompasses human antibodies with different Vregions. It is within the skill of one in the art to determine suitableV region sequences and to optimize these sequences. Methods forobtaining antibodies with reduced immunogenicity are also described inU.S. Pat. No. 5,270,202 and EP 699,755.

In certain applications, such as when an antigen-binding fragment or DCAis expressed in a suitable storage medium such as a plant seed, theantigen-binding fragment can be stored without purification. Fiedler etal. (1995) Biotechnol. 13:1090-1093. For most applications, it isgenerally preferable that the polypeptide is at least partially purifiedfrom other cellular constituents. Preferably, the peptide is at leastabout 50% pure as a weight percent of total protein. More preferably,the peptide is at least about 50-75% pure. For clinical use, the peptideis preferably at least about 80% pure.

If the peptides are to be administered to an individual, preferably itis at least 80% pure, more preferably at least 90% pure, even morepreferably at least 95% pure and free of pyrogens and othercontaminants. In this context, the percent purity is calculated as aweight percent of the total protein content of the preparation, and doesnot include constituents which are deliberately added to the compositionpurification.

The invention also encompasses methods of detecting, enumerating and/oridentifying DCs and subsets thereof, in a biological sample andmeasuring antigens such as soluble BDCA-2, BDCA-3 or BDCA-4 and/or DCsin body fluids. The methods include obtaining a biological sample,contacting the sample with an antigen-binding fragment described hereinunder conditions that allow antibody-antigen-binding and detectingbinding, if any, of the antibody to the sample as compared to a control,biological sample.

After a biological sample is suitably prepared, for instance byenriching for DC concentration or antigen concentration, it is mixedwith excess antigen-binding fragments under conditions that permitformation of a complex between DCs or antigen and the antibody. Theamount of complex formed or the number of complex bearing DCs thendetermined, and eventually compared with complexes formed with standardsamples containing known amounts of target antigen in the range expectedor known DC concentrations. Complex formation can be observed byimmunoprecipitation or nephelometry, but it is generally more sensitiveto employ a reagent labeled with such labels as radioisotopes like ¹²⁵I,enzymes like peroxidase and β-galactosidase, or fluorochromes likefluorescein. Methods of detecting cells and antigens are well known inthe art. For cell detection, flow cytometry is particularly useful, withantigen, ELISA is preferred.

The specific recognition of an anti-DC antigen-binding fragment to anantigen can be tested by any immunoassay known in the art. Any form ofdirect binding assay is suitable. In one such assay, one of the bindingpartners, the antigen or the putative antigen-binding fragment, islabeled. Suitable labels include, but are not limited to, radioisotopessuch as ¹²⁵I, enzymes such as peroxidase, fluorescent labels such asfluorescein, and chemiluminescent labels. Typically, the other bindingpartner is insolubilized (for example, by coating onto a solid phasesuch as a microtiter plate) to facilitate-removal of unbound solublebinding partner. After combining the labeled binding partner with theunlabeled binding partner, the solid phase is washed and the amount ofbound label is determined.

When used for immunotherapy, the antigen-binding fragments describedherein can be unlabeled or labeled with a therapeutic agent as describedherein and as known in the art. These agents can be coupled eitherdirectly or indirectly to the antigen-binding fragments of theinvention. One example of indirect coupling is by use of a spacermoiety. These spacer moieties, in turn, can be either insoluble orsoluble (Diener et al. (1986) Science 231:148) and can be selected toenable drug release at the target site. Examples of therapeutic agentsthat can be coupled to antigen-binding fragments for immunotherapyinclude, but are not limited to, antigens, including tumor antigens,viral antigens, bacterial antigens, parasite-derived antigens andautoantigens, bioresponse modifiers, drugs, radioisotopes, lectins, andtoxins. Bioresponse modifiers include cytokines and chemokines whichinclude, but are not limited to, IL-2, IL-3, IL-4, G-CSF, GM-CSF, IL-10,IL-12, TGF-β, MTP-AB, SDF-1, Lymphotactin, DC-CK1, Eotoxins, IP-10,TARC, Rantes, MIP-1α, MIP-1β, SLC, ITAC, MIE, MDC, MCP-1, TCA-3, MCP-2,-3, -4 and interferons. Interferons with which antigen-binding fragmentscan be labeled include IFN-α, IFN-β, and IFN-γ and their subtypes.

In using radioisotopically conjugated antigen-binding fragments forimmunotherapy, certain isotypes can be more preferable than othersdepending on such factors as isotype stability and emission. If desired,cell population recognition by the antigen-binding fragment can beevaluated by the in vivo diagnostic techniques described below. Ingeneral, α and β particle-emitting radioisotopes are preferred inimmunotherapy. For example, a high energy β emitter capable ofpenetrating several millimeters of tissue, such as ⁹⁰Y, can bepreferable. On the other hand, a short range, high energy α emitter,such as ²¹²Bi, can be preferable. Examples of radioisotopes which can bebound to the antigen-binding fragments of the invention for therapeuticpurposes include, but are not limited to, ¹²⁵I, ¹³¹ I, ⁹⁰Y, 67Cu, ²¹²Bi,²¹¹At, ₂₁₂Pb, ⁴⁷Sc, ¹⁰⁹Pd, and ¹⁸⁸Re.

Lectins are proteins, usually isolated from plant material, which bindto specific sugar moieties. Many lectins are also able to agglutinatecells and stimulate lymphocytes. However, ricin is a toxic lectin whichhas been used immunotherapeutically. This is preferably accomplished bybinding the α peptide chain of ricin, which is responsible for toxicity,to the antibody molecule to enable site specific delivery of the toxiceffect.

Toxins are poisonous substances produced by plants, animals, ormicroorganisms that, in sufficient dose, are often lethal. Diphtheriatoxin is a substance produced by Corynebacterium diphtheria which can beused therapeutically. This toxin consists of an α and β subunit whichunder proper conditions can be separated. The toxic A chain componentcan be bound to an antigen-binding fragment described herein and usedfor site specific delivery to a specific subset of DCs.

Recombinant methods are well known in the art. The practice of theinvention employs, unless otherwise indicated, conventional techniquesof molecular biology (including recombinant techniques), microbiology,cell biology, biochemistry and immunology, which are within the skill ofthe art. Such techniques are explained fully in the literature, such as,“Molecular Cloning: A Laboratory Manual”, second edition (Sambrook etal., 1989); “Oligonucleotide Synthesis” (Gait, ed., 1984); “Animal CellCulture” (Freshney, ed., 1987); “Methods in, Enzymology” (AcademicPress, Inc.); “Handbook of Experimental Immunology” (Wei & Blackwell,eds.); “Gene Transfer Vectors for Mammalian Cells” (Miller & Calos,eds., 1987); “Current Protocols in Molecular Biology” (Ausubel et al.,eds., 1987); “PCR: The Polymerase Chain Reaction”, (Mullis et al., eds.,1994); and “Current Protocols in Immunology” (Coligan et al., eds.,1991). These techniques are applicable to the production of thepolynucleotides and polypeptides, and, as such, can be considered inmaking and practicing the invention. Particularly useful techniques arediscussed in the sections that follow.

The invention provides various polynucleotides encoding BDCA antigens.The invention also encompasses polynucleotides encoding for functionallyequivalent variants and derivatives of these antigens and functionallyequivalent fragments thereof that can enhance, decrease or notsignificantly affect properties of the polypeptides encoded thereby.These functionally equivalent variants, derivatives, and fragments maydisplay the ability to specifically bind to their respective antibodies.For instance, changes that will not significantly affect properties ofthe encoded polypeptide include, but are not limited to changes in a DNAsequence that do not change the encoded amino acid sequence, as well asthose that result in conservative substitutions of amino acid residues,one or a few amino acid residue deletions or additions, and substitutionof amino acid residues by amino acid analogs. Conservative substitutionsare those which conservative amino acid substitutions areglycine/alanine; valine/isoleucine/leucine; asparagine/glutamine;aspartic acid/glutamic acid; serine/threonine/methionine;lysine/arginine; and phenylalanine/tyrosine/tryptophan.

The polynucleotides of the invention can comprise additional sequences,such as additional encoding sequences within the same transcriptionunit, controlling elements such as promoters, ribosome binding sites,and polyadenylation sites, additional transcription units under controlof the same or a different promoter, sequences that permit cloning,expression, and transformation of a host cell, and any such construct ascan be desirable to provide embodiments of this invention.

The invention encompasses a polynucleotide of at least about 15consecutive nucleotides, preferably at least about 20 nucleotides, morepreferably at least about 25 consecutive nucleotides, more preferably atleast about 35 consecutive nucleotides, more preferably at least about50 consecutive nucleotides, even more preferably at least about 75nucleotides, still more preferably at least about 100 nucleotides, stillmore preferably at least about 200 nucleotides, and even more preferablyat least about 300 nucleotides that forms a stable hybrid with apolynucleotide encoding BDCA-2 and BDCA-3, preferably the cDNA sequencefound in FIG. 12. Any set of conditions can be used for this test,provided at least one set exists where the test polynucleotidedemonstrates the required specificity.

Hybridization reactions can be performed under conditions of different“stringency”. Conditions that increase stringency of a hybridizationreaction are published. See, for example, Sambrook and Maniatis.Examples of relevant conditions include (in order of increasingstringency): incubation temperatures of 25° C., 37° C., 50° C. and 68°C.; buffer concentrations of 10×SSC, 6×SSC, 1×SSC, 0.1×SSC (where SSC is0.15 M NaCl and 15 mM citrate buffer) and their equivalent using otherbuffer systems; formamide concentrations of 0%, 25%, 50%, and 75%;incubation times from minutes to 24 hours; 1, 2, or more washing steps;wash incubation times of 11, 2, or 15 minutes; and wash solutions of6×SSC, 1×SSC, 0.1×SSC, or deionized water.

The invention also provides polynucleotides encoding the BDCApolypeptides. Preferably, the polypeptides are or are derived from thosein FIG. 12.

The invention also provides polynucleotides covalently linked with adetectable label. Such polynucleotides are useful, for example, asprobes for detection of related nucleotide sequences.

The polynucleotides of this invention can be obtained using chemicalsynthesis, recombinant cloning methods, PCR, or any combination thereof.Methods of chemical polynucleotide synthesis are well known in the artand need not be described in detail herein. One of skill in the art canuse the sequence data provided herein to obtain a desired polynucleotideby employing a DNA synthesizer or ordering from a commercial service.

Alternatively, nucleotides encoding BDCAs and the peptides encodedthereby can be obtained from a producing cell line, cloning vector, orexpression vector. RNA or DNA encoding the desired sequence can beisolated, amplified, and processed by standard recombinant techniques.Such techniques include digestion with restriction nucleases, andamplification by polymerase chain reaction (PCR), or a suitablecombination thereof. PCR technology is described in U.S. Pat. Nos.4,683,195; 4,800,159; 4,754,065; and 4,683,202, as well as PCR: ThePolymerase Chain Reaction, Mullis et al. eds., Birkauswer Press, Boston(1994). Isolation and purification of the peptides encoded thereby canbe by any method known in the art.

Polynucleotides comprising a desired sequence can be inserted into asuitable vector, the vector in turn can be introduced into a suitablehost cell for replication and amplification. Polynucleotides can beinserted into host cells by any means known in the art. Cells aretransformed by introducing an exogenous polynucleotide by direct uptake,endocytosis, transfection, f-mating or electroporation. Once introduced,the exogenous polynucleotide can be maintained within the cell as anon-integrated vector (such as a plasmid) or integrated into the hostcell genome. Amplified DNA can be isolated from the host cell bystandard methods. See, e.g., Sambrook et al. (1989). RNA can also beobtained from transformed host cell, it can be obtained by using aDNA-dependent RNA polymerase.

The present invention further includes a variety of vectors comprising apolynucleotide encoding BDCA-2 and/or BDCA-3. These vectors can be usedfor expression of recombinant polypeptides as well as a source ofBDCA-encoding polynucleotides. Cloning vectors can be used to obtainreplicate copies of the polynucleotides, or for storing thepolynucleotides in a depository for future recovery. Expression vectors(and host cells containing these expression vectors) can be used toobtain polypeptides produced from the polynucleotides they contain. Theycan also be used where it is desirable to express BDCA-2 and/or BDCA-3in an individual and thus have intact cells capable of synthesizing thepolypeptide, such as in gene therapy. Suitable cloning and expressionvectors include any known in the art e.g., those for use in bacterial,mammalian, yeast and insect expression systems. Specific vectors andsuitable host cells are known in the art and are not described in detailherein. See e.g. Gacesa and Ramji, Vectors, John Wiley & Sons (1994).

Cloning and expression vectors typically contain a selectable marker(for example, a gene encoding a protein necessary for the survival orgrowth of a host cell transformed with the vector), although such amarker gene can be carried on another polynucleotide sequenceco-introduced into the host cell. Only those host cells into which aselectable gene has been introduced will grow under selectiveconditions. Typical selection genes either: (a) confer resistance toantibiotics or other toxic substances, e.g., ampicillin, neomycin,methotrexate; (b) complement auxotrophic deficiencies; or (c) supplycritical nutrients not available from complex media. The choice of theproper marker gene will depend on the host cell, and appropriate genesfor different hosts are known in the art. Vectors also typically containa replication system recognized by the host.

Suitable cloning vectors can be constructed according to standardtechniques, or can be selected from a large number of cloning vectorsavailable in the art. While the cloning vector selected can varyaccording to the host cell intended to be used, useful cloning vectorswill generally have the ability to self-replicate, can possess a singletarget for a particular restriction endonuclease, or can carry genes fora marker that can be used in selecting clones containing the vector.Suitable examples include plasmids and bacterial viruses, e.g., pUC18,mp18, mp19, pBR322, pMB9, ColE1, pCR1, RP4, phage DNAs, and shuttlevectors such as pSA3 and pAT28. These and many other cloning vectors areavailable from commercial vendors such as BioRad, Stratagene, andInvitrogen.

Expression vectors generally are replicable polynucleotide constructsthat contain a polynucleotide encoding a BDCA of interest. Thepolynucleotide encoding BDCA is operatively linked to suitabletranscriptional controlling elements, such as promoters, enhancers andterminators. For expression (i.e., translation), one or moretranslational controlling elements are also usually required, such asribosome binding sites, translation initiation sites, and stop codons.These controlling elements (transcriptional and translational) can bederived from a gene encoding a BDCA, or they can be heterologous (i.e.,derived from other genes or other organisms). A polynucleotide sequenceencoding a signal peptide can also be included to allow a BDCA to crossor lodge in cell membranes or be secreted from the cell. A number ofexpression vectors suitable for expression in eukaryotic cells includingyeast, avian, and mammalian cells are known in the art. One example ofan expression vector is pcDNA3 (Invitrogen, San Diego, Calif.), in whichtranscription is driven by the cytomegalovirus (CMV) earlypromoter/enhancer. This vector also contains recognition sites formultiple restriction enzymes for insertion of the polynucleotide ofinterest. Another example of an expression vector (system) is thebaculovirus/insect system. Other suitable for use in antibody-targetedgene therapy comprising a polynucleotide encoding a BDCA. Suitablesystems are described for instance by Brown et al. (1994) Virol.198:477-488; and Miyamura et al. (1994) Proc. Natl. Acad. Sci. USA91:8507-8511.

The vectors containing the polynucleotides of interest can be introducedinto the host cell by any of a number of appropriate means, includingelectroporation, transfection employing calcium chloride, rubidiumchloride, calcium phosphate, DEAE-dextran, or other substances;microprojectile bombardment; lipofection; and infection. The choice ofmeans of introducing vectors or polynucleotides encoding BDCAs willoften depend on features of the on the host cell.

Once introduced into a suitable host cell, expression of a BDCA can bedetermined using any assay known in the art. For example, the presencethereof can be detected by RIA or ELISA of the culture supernatant (ifthe polypeptide is secreted) or cell lysates.

A vector of this invention can contain one or more polynucleotidesencoding a BDCA. It can also contain polynucleotide sequences encodingother polypeptides that enhance, facilitate, or modulate the desiredresult, such as cytokines, including, but not limited to, IL-2, IL-4,GM-CSF, TNF-α and IFN-γ. Also embodied in this invention are vacciniavectors encoding for recombinant BDCAs.

Other embodiments of this invention are host cells transformed withpolynucleotides encoding BDCAs and vectors comprising the polynucleotidesequences, as described above. Both prokaryotic and eukaryotic hostcells can be used. Prokaryotic hosts include, but are not limited to,bacterial cells, for example E. coli and mycobacteria. Eukaryotic hostsinclude, but are not limited to, yeast, insect, avian, plant andmammalian cells. Host systems are known in the art and need not bedescribed in detail herein. Examples of a mammalian host cells include,but are not limited to, CHO and NS0, obtainable from the EuropeanCollection of Cell Cultures (England). Transfection of NS0 cells with aplasmid, for example, which is driven by a CMV promoter, followed byamplification of this plasmid in using glutamine synthetase provides auseful system for protein production. Cockett et al. (1990)Bio/Technology 8:662-667.

The host cells of this invention can be used, inter alia, asrepositories of polynucleotides encoding BDCAs, or as vehicles forproduction thereof. They can be used also as vehicles for in vivoexpression of BDCAs.

The polynucleotides of this invention have several uses. They areuseful, for example, in expression systems for the production of BDCA.They are also useful as hybridization probes to assay for the presenceof polynucleotides encoding BDCAs or related sequences in a sample usingmethods well known to those in the art. Further, the polynucleotides arealso useful as primers to effect amplification of desiredpolynucleotides. The polynucleotides of this invention are also usefulin pharmaceutical compositions including vaccines and for gene therapy.

The polynucleotides can also be used as hybridization probes fordetection of BDCA-encoding sequences. Suitable hybridization samplesinclude cells transformed ex vivo for use in gene therapy. In oneillustration, DNA or RNA is extracted from a sample, and optionally runon a gel and/or digested with restriction nucleases. The processedsample polynucleotide is typically transferred to a medium suitable forwashing. The sample polynucleotide is then contacted with theBDCA-encoding polynucleotide probe under conditions that permit a stableduplex to form if the sample contains a complementary polynucleotidesequence. Any stable duplexes formed are detected by any suitable means.For example, the polynucleotide probe can be supplied in labeled form,and label remaining with the sample after washing will directly reflectthe amount of stable duplex formed. In a second illustration,hybridization is performed in situ. A suitably prepared tissue sample isoverlaid with a labeled probe to indicate the location of BDCA-encodingsequences.

A short polynucleotide can also be used as a primer for a PCR reaction,particularly to amplify a longer sequence comprising a regionhybridizing with the primer. This can be conducted preparatively, inorder to produce polynucleotide for further genetic manipulation. It canalso be conducted analytically, to determine whether a BDCA-encodingpolynucleotide is present, for example, in a sample of diagnosticinterest.

Another use of the polynucleotides is in vaccines and gene therapy. Thegeneral principle is to administer the polynucleotide so that it eitherpromotes or attenuates the expression of the polypeptide encodedthereby. Thus, the present invention includes methods of inducing animmune response and methods of treatment comprising administration of aneffective amount of polynucleotides encoding BDCAs to an individual. Inthese methods, a polynucleotide encoding BDCA is administered to anindividual, either directly or via cells transfected with thepolynucleotide. Preferably, the polynucleotide is in the form of acircular plasmid, preferably in a supercoiled configuration. Preferably,the polynucleotide is replicated inside a cell. Thus, the polynucleotideis operatively linked to a suitable promoter, such as a heterologouspromoter that is intrinsically active in cells of the target tissuetype. Preferably, once in cell nuclei, plasmids persist as circularnon-replicating episomal molecules. In vitro mutation can be carried outwith plasmid constructs to encode, for example, molecules with greateraffinity and/or avidity.

To determine whether plasmids containing BDCA polynucleotides arecapable of expression in eukaryotic cells, cells such as COS-7, CHO, orHeLa can be transfected with the plasmids. Expression is then determinedby immunoassay; for example, by Western blot. Smaller BDCAs can bedetected, for example, by constructing the plasmid so that the resultantpolypeptide is fused with a tag, such as a target epitope or enzymelabel. Further characterization of the expressed polypeptide can beachieved by purifying the peptide and then conducting one of thefunctional assays described herein.

In one mode of gene therapy, the polynucleotides of this invention areused for genetically altering cells ex vivo. In this strategy, cellsremoved from a donor or obtained from a cell line are transfected ortransduced with BDCA vectors, and then administered to a recipient.Suitable cells for transfection include peripheral blood mononuclearcells.

In another mode of gene therapy, the polynucleotides of this inventionare used for genetically altering cells in vivo. The purpose caninclude, but is not limited to, treating various types of cancer.

The polynucleotides can also be used to produce cells that do notexpress BDCA-2, and transgenic animals expressing BDCA-2.

Also obtained from the invention are cells engineered not to express orto express at significantly reduced levels BDCA-2. Such cells may beproduced by selecting a cell, preferably a DC, and providing to the cellan expression construct comprising a polynucleotide encoding a BDCA-2gene wherein the polynucleotide is positioned antisense to andoperatively linked to a promoter. The expression of such apolynucleotide effectively produces a cell deficient in BDCA-2.

In other embodiments the present invention provides a method for thepreparation of recombinant host cells that produce significantly reducedamounts or even “knockout” the production of BDCA-2. These recombinanthost cells can be prepared by using one or more means that are wellknown to those of skill in the art. For example, gene expression can beinhibited by the incorporation of constructs for antisense DNA or RNAinto the genome. Deletions or mutations of the endogenous BDCA-2 genescan render them nonfunctional. Nucleic acids encodingribozymes-RNA-cleaving enzymes—that specifically cleave BDCA-2 mRNA canbe introduced into the recombinant host cells.

The term “knockout” refers to partial or complete suppression of theexpression of at least a portion of a protein encoded by an endogenousDNA sequence, e.g. BDCA-2, in a cell. The term “knockout construct”refers to a nucleic acid sequence that is designed to decrease orsuppress expression of a protein encoded by endogenous DNA sequences ina cell.

These recombinant constructs can be incorporated into knockout mammalssuch that the production of BDCA-2 is suppressed in DCs. The preparationof knock out and transgenic animals is well known to those of skill inthe art and is described in 5,434,340, 5,530,179 and 5,557,032.

The invention further provides methods for producing animals and theanimals so produced that over-express BDCA-2. These methods generallycomprise introducing animal cells into an animal, the animal cellshaving been treated in vitro to insert therein a DNA segment encoding aBDCA-2 polypeptide, the animal cells expressing in vivo in the animalBDCA-2.

D. Kits

The invention encompasses kits containing anti-DC-specificantigen-binding fragments, for measuring BDCA-2 including solubleBDCA-2, including isoforms thereof, in serum and other sources.Diagnostic procedures using the kits can be performed by diagnosticlaboratories, experimental laboratories, practitioners, or privateindividuals. The clinical sample is optionally pre-treated forenrichment of the target being tested for. The user then applies areagent contained in the kit in order to detect the changed level oralteration in the diagnostic component.

Optionally, the reagent can be conjugated with a label to permitdetection of any complex formed with the target in the sample. Inanother option, a second reagent is provided that is capable ofcombining with the first reagent after it has found its target andthereby supplying the detectable label. For example, labeled anti-murineIgG can be provided as a secondary reagent. Labeled avidin is asecondary reagent when the primary reagent has been conjugated tobiotin.

The kits can be employed on a variety of biological samples including,both liquid samples, cell suspensions and tissue samples. Suitableassays that can be supplied in kit form include those described herein.

Each reagent is supplied in a solid form or dissolved/suspended in aliquid buffer suitable for inventory storage and later for exchange oraddition into the reaction medium when the test is performed. Suitablepackaging is provided. The kit can optionally provide additionalcomponents that are useful in the procedure. These optional componentsinclude, but are not limited to, buffers, capture reagents, developingreagents, labels, reacting surfaces, means for detection, controlsamples, instructions, and interpretive information.

E. Therapeutic Compositions 1. Compositions of Matter

The preparation of pharmaceutical compositions described herein isconducted in accordance with generally accepted procedures for thepreparation of pharmaceutical preparations. See, for example,Remington's Pharmaceutical Sciences 18th Edition (1990), E. W. Martined., Mack Publishing Co., PA. Depending on the intended use and mode ofadministration, it can be desirable to process the active ingredientfurther in the preparation of pharmaceutical compositions. Appropriateprocessing can include sterilizing, mixing with appropriate non-toxicand non-interfering components, dividing into dose units, and enclosingin a delivery device. In one embodiment, the therapeutic compositionscontain DCs, subpopulations thereof or mixtures thereof. In anotherembodiment, the compositions contain the antigen-binding fragmentsdescribed herein. Preferably, the antigen-binding fragments are, or arederived from, the mAbs listed in Table 1. Preferably the DC compositionscontain DCs isolated with one of these antigen-binding fragments.

(a) General Modes of Administration

Pharmaceutical compositions of the invention are administered by a modeappropriate for the form of composition. Typical routes includeintravenous, subcutaneous, intramuscular, intraperitoneal, intradermal,oral, intranasal, intradermal, and intrapulmonary (i.e., by aerosol).Pharmaceutical compositions for human use are typically administered bya parenteral route, most typically intravenous, subcutaneous,intramuscular. Although not required, pharmaceutical compositions arepreferably supplied in unit dosage form suitable for administration of aprecise amount. Also contemplated by this invention are slow release orsustained release forms, whereby a relatively consistent level of theactive compound are provided over an extended period.

(b) Liquid Formulations

Liquid pharmaceutically acceptable compositions can, for example, beprepared by dissolving or dispersing a polypeptide or polynucleotideembodied herein in a liquid excipient, such as water, saline, aqueousdextrose, glycerol, or ethanol. The composition can optionally alsocontain other medicinal agents, pharmaceutical agents, carriers, andauxiliary substances such as wetting or emulsifying agents, and pHbuffering agents. Compositions for injection can be supplied as liquidsolutions or suspensions, as emulsions, or as solid forms suitable fordissolution or suspension in liquid prior to injection.

Pharmaceutical compositions for oral, intranasal, or topicaladministration can be supplied in solid, semi-solid or liquid forms,including tablets, capsules, powders, liquids, and suspensions. Foradministration via the respiratory tract, a preferred composition is onethat provides a solid, powder, or liquid aerosol when used with anappropriate aerosolizer device.

The invention also encompasses compositions comprising liposomes withmembrane bound peptide to specifically deliver the liposome to the areaof the tumor or neoplastic cells or to the immune system. Theseliposomes can be produced such that they contain, in addition topeptide, immunotherapeutic agents such as those described above whichwould then be released at the recognition site. Wolff et al. (1984)Biochem. Biophys. Acta 802:259. Another such delivery system utilizeschimeric parvovirus B19 capsids for presentation of the antigen-bindingfragments. Brown et al. (1994) Virol. 198:477-488; and Miyamura et al.(1994) Proc. Natl. Acad. Sci. USA 91:8507-8511. Such chimeric systemsare encompassed for use herein.

Compositions embodied in this invention can be assessed for theirefficacy in a number of ways. Accordingly, test compounds are preparedas a suitable pharmaceutical composition and administered to testsubjects. Initial studies are preferably done in small animals such asmice or rabbits, optionally next in non-human primates and thenultimately in humans. Immunogenicity is preferably tested in individualswithout a previous antibody response. A test composition in anappropriate test dose is administered on an appropriate treatmentschedule. It can be appropriate to compare different doses and scheduleswithin the predicted range. The dosage ranges for the administration ofantigen-binding fragments are large enough to produce the desired effectin which the symptoms of the disease are ameliorated without causingundue side effects such as unwanted cross-reactions and anaphylacticreactions. Generally, the dosage will vary with the age, condition, sexand extent of the disease in the patient and can be determined by one ofskill in the art. The dosage can be adjusted by the individual physicianin the event of any complication. Generally, when the compositions areadministered conjugated with therapeutic agents, lower dosages,comparable to those used for in vivo immunodiagnostic imaging, can beused.

2. Antigen-Binding Fragments

The invention encompasses pharmaceutical compositions containing theantigen-binding fragments described herein. Such pharmaceuticalcompositions are useful for inducing or aiding an immune response andtreating neoplastic diseases, or including tolerance and treatingautoimmune diseases, (GvHD, allograft rejection, allergen, etc.) eitheralone or in conjunction with other forms of therapy, such aschemotherapy, radiotherapy or immune therapies described in WO98/23735;WO98/34642; WO97/10000; WO97/10001; and WO97/06821. Other methods oftreatment are described herein and/or known in the art. Suitablediseases include, without limitation, viral, parasitic, bacterial,fungal, neoplastic and autoimmune.

In a murine breast cancer model, Flt3-Ligand (Flt3-L), a stimulatorycytokine for a variety of hematopoietic lineages, including DCs and Bcells, has been used in conjunction with murine breast cancer cells as avaccine. Chen et al. (1997) Cancer Res. 57:3511-6. DCs can also beloaded with or transduced to express tumor antigens; these cells arethen used as adjuvants to tumor vaccination. DCs presenttumor-associated antigens endogenously to the afferent lymphatic systemin the appropriate MHC-restricted context. Wan et al. (1997) Hum. GeneTher. 8:1355-63; Peiper et al. (1997) Surgery 122:235-41; and Smith etal. (1997) Int. Immunol. 9:1085-93. Current melanoma vaccines manipulateantigen presentation networks and combine the best cellular and antibodyanti-tumor immune response effective in mediating tumor protectiveimmunity. These therapies have caused regression, delayed diseaseprogression or an improvement in survival in some cases, with a paucityof side effects. Kuhn et al. (1997) Dermatol. Surg. 23:649-54. Melanomavaccines are also reviewed in Conforti et al. (1997) J. Surg. Oncol.66:55-64.

Vaccines can be packaged in pharmaceutically acceptable carriers,admixed with adjuvants or other components (such as cytokines) as knownin the art. Vaccines for veterinarian use are substantially similar tothat in humans with the exception that adjuvants containing bacteria andbacterial components such as Freund's complete or incomplete adjuvants,are allowed in the formulations.

F. Methods of Treatment

Also included in this invention are methods for treating a variety ofdisorders as described herein and/or known in the art. The methodscomprise administering an amount of a pharmaceutical compositioncontaining a composition of the invention in an amount effective toachieve the desired effect, be it palliation of an existing condition orprevention of recurrence. For treatment of cancer, the amount of apharmaceutical composition administered is an amount effective inproducing the desired effect. An effective amount can be provided in oneor a series of administrations. An effective amount can be provided in abolus or by continuous perfusion. Suitable active agents include theanti-neoplastic drugs, bioresponse modifiers and effector cells such asthose described by Douillard et al. (1986) Hybridomas (Supp. 1:5139).

Pharmaceutical compositions and treatment modalities are suitable fortreating a patient by either directly or indirectly eliciting an immuneresponse against neoplasia. An “individual,” “patient” or “subject” is avertebrate, preferably a mammal, more preferably a human. Mammalsinclude, but are not limited to: humans, wild animals, feral animals,farm animals, sport animals, and pets. A “cancer subject” is a mammal,preferably a human, diagnosed as having a malignancy or neoplasia or atrisk thereof.

As used herein, “treatment” refers to clinical intervention in anattempt to alter the disease course of the individual or cell beingtreated, and can be performed either for prophylaxis or during thecourse of clinical pathology. Therapeutic effects of treatment includewithout limitation, preventing occurrence or recurrence of disease,alleviation of symptoms, diminishment of any direct or indirectpathological consequences of the disease, preventing metastases,decreasing the rate of disease progression, amelioration or palliationof the disease state, and remission or improved prognosis.

The “pathology” associated with a disease condition is any conditionthat compromises the well-being, normal physiology, or quality of lifeof the affected individual. This can involve, but is not limited to,destructive invasion of affected tissues into previously unaffectedareas, growth at the expense of normal tissue function, irregular orsuppressed biological activity, aggravation or suppression of aninflammatory or immunologic response, increased susceptibility to otherpathogenic organisms or agents, and undesirable clinical symptoms suchas pain, fever, nausea, fatigue, mood alterations, and such otherdisease-related features as can be determined by an attending physician.

An “effective amount” is an amount sufficient to effect a beneficial ordesired clinical result upon treatment. An effective amount can beadministered to a patient in one or more doses. In terms of treatment,an effective amount is an amount that is sufficient to palliate,ameliorate, stabilize, reverse or slow the progression of the disease,or otherwise reduce the pathological consequences of the disease. Theeffective amount is generally determined by the physician on acase-by-case basis and is within the skill of one in the art. Severalfactors are typically taken into account when determining an appropriatedosage to achieve an effective amount. These factors include age, sexand weight of the patient, the condition being treated, the severity ofthe condition and the form and effective concentration of theantigen-binding fragment administered.

The term “immunomodulatory” or “modulating an immune response” as usedherein includes immunostimulatory as well as immunosuppressive effects.Immunostimulatory effects include, but are not limited to, those thatdirectly or indirectly enhance cellular or humoral immune responses.Examples of immunostimulatory effects include, but are not limited to,increased antigen-specific antibody production; activation orproliferation of a lymphocyte population such as NK cells, CD4⁺ cells,CD8⁺ cells, macrophages and the like; increased synthesis of cytokinesor chemokines including, but not limited to, IL-1, IL-2, IL-4, IL-5,IL-6, IL-12, interferons, TNF-α, IL-10, TGF-β and the like.Immunosuppressive effects include those that directly or indirectlydecrease cellular or humoral immune responses. Examples ofimmunosuppressive effects include, but are not limited to, a reductionin antigen-specific antibody production such as reduced IgE production;activation of lymphocyte or other cell populations that haveimmunosuppressive activities such as those that result in immunetolerance; and increased synthesis of cytokines that have suppressiveeffects toward certain cellular functions including, but not limited toIL-10 and TGF-β. One example of this is IFN-γ, which appears to blockIL-4 induced class switch to IgE and IgG1, thereby reducing the levelsof these antibody subclasses.

Suitable human subjects for cancer therapy further comprise twotreatment groups, which can be distinguished by clinical criteria.Patients with “advanced disease” or “high tumor burden” are those whobear a clinically measurable tumor. A clinically measurable tumor is onethat can be detected on the basis of tumor mass (e.g., by palpation, CATscan, sonogram, mammogram or X-ray; positive biochemical orhistopathologic markers on their own are insufficient to identify thispopulation). A pharmaceutical composition embodied in this invention isadministered to these patients to elicit an anti-tumor response, withthe objective of palliating their condition. Ideally, reduction in tumormass occurs as a result, but any clinical improvement constitutes abenefit. Clinical improvement includes decreased risk or rate ofprogression or reduction in pathological consequences of the tumor.

A second group of suitable subjects is known in the art as the “adjuvantgroup.” These are individuals who have had a history of cancer, but havebeen responsive to another mode of therapy. The prior therapy can haveincluded (but is not restricted to, surgical resection, radiotherapy,and traditional chemotherapy. As a result, these individuals have noclinically measurable tumor. However, they are suspected of being atrisk for progression of the disease, either near the original tumorsite, or by metastases.

“Adjuvant” as used herein has several meanings, all of which will beclear depending on the context in which the term is used. In the contextof a pharmaceutical preparation, an adjuvant is a chemical or biologicalagent given in combination (whether simultaneously or otherwise) with,or recombinantly fused to, an antigen to enhance immunogenicity of theantigen. For review see, Singh et al. (1999) Nature Biotech.17:1075-1081. Isolated DCs have also been suggested for use asadjuvants. Compositions for use therein are included in this invention.In the context of cancer diagnosis or treatment, adjuvant refers to aclass of cancer patients with no clinically detectable tumor mass, butwho are suspected of risk of recurrence.

This group can be further subdivided into high-risk and low-riskindividuals. The subdivision is made on the basis of features observedbefore or after the initial-treatment. These features are known in theclinical arts, and are suitably defined for each different cancer.Features typical of high-risk subgroups are those in which the tumor hasinvaded neighboring tissues, or who show involvement of lymph nodes.

Another suitable group is those with a genetic predisposition to cancerbut who have not yet evidenced clinical signs of cancer. For instance,women testing positive for a genetic mutation associated with breastcancer, but still of childbearing age, can wish to receive one or moreof the antigen-binding fragments described herein in treatmentprophylactically to prevent the occurrence of cancer until it issuitable to perform preventive surgery.

Human cancer patients, including, but not limited to, glioblastoma,melanoma, neuroblastoma, adenocarcinoma, glioma, soft tissue sarcoma,and various carcinomas (including small cell lung cancer) are especiallyappropriate subjects. Suitable carcinomas further include any known inthe field of oncology, including, but not limited to, astrocytoma,fibrosarcoma, myxosarcoma, liposarcoma, oligodendroglioma, ependymoma,medulloblastoma, primitive neural ectodermal tumor (PNET),chondrosarcoma, osteogenic sarcoma, pancreatic ductal adenocarcinoma,small and large cell lung adenocarcinomas, chordoma, angiosarcoma,endotheliosarcoma, squamous cell carcinoma, bronchoalveolarcarcinoma,epithelial adenocarcinoma, and liver metastases thereof,lymphangiosarcoma, lymphangioendotheliosarcoma, hepatoma,cholangiocarcinoma, synovioma, mesothelioma, Ewing's tumor,rhabdomyosarcoma, colon carcinoma, basal cell carcinoma, sweat glandcarcinoma, papillary carcinoma, sebaceous gland carcinoma, papillaryadenocarcinoma, cystadenocarcinoma, medullary carcinoma, bronchogeniccarcinoma, renal cell carcinoma, bileduct carcinoma, choriocarcinoma,seminoma, embryonal carcinoma, Wilms' tumor, testicular tumor,medulloblastoma, craniopharyngioma, ependymoma, pinealoma,hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma,neuroblastoma, retinoblastoma, leukemia, multiple myeloma, Waldenstrom'smacroglobulinemia, and heavy chain disease, breast tumors such as ductaland lobular adenocarcinoma, squamous and adenocarcinomas of the uterinecervix, uterine and ovarian epithelial carcinomas, prostaticadenocarcinomas, transitional squamous cell carcinoma of the bladder, Band T cell lymphomas (nodular and diffuse) plasmacytoma, acute andchronic leukemias, malignant melanoma, soft tissue sarcomas andleiomyosarcomas.

The patients can have an advanced form of disease, in which case thetreatment objective can include mitigation or reversal of diseaseprogression, and/or amelioration of side effects. The patients can havea history of the condition, for which they have been treated, in whichcase the therapeutic objective will typically include a decrease ordelay in the risk of recurrence.

Autoimmune disorders are the caused by a misdirected immune responseresulting in self-destruction of a variety of cells, tissues and organs.The cause of these disorders is unknown. Recognition of self through theMHC is known to be of importance in an immune response. However,prevention of an autoimmune response and the cells responsible forautoimmunity are not well understood.

Autoimmunity results from a combination of factors, including genetic,hormonal, and environmental influences. Many autoimmune disorders arecharacterized by B cell hyperactivity, marked by proliferation of Bcells and autoantibodies and by hypergammaglobulinemia. B cellhyperactivity is probably related to T cell abnormalities. Hormonal andgenetic factors strongly influence the incidence of autoimmunedisorders; for example, lupus erythematosus predominantly affects womenof child-bearing age, and certain HLA haplotypes are associated with anincreased risk of specific autoimmune disorders.

Common autoimmune disorders include, but are not limited to, rheumatoidarthritis, juvenile rheumatoid arthritis, psoriatic arthritis,ankylosing spondylitis, Sjögren's syndrome, lupus erythematosus,Goodpasture's syndrome, Reiter's syndrome, scleroderma, vasculitis,polymyositis and dermatomyositis. Many of these conditions includeaberrant inflammatory reactions related to the immunologic disorders.The DCs described herein are suitable for use in treatment of thesedisorders particularly when used to inactivate or inducetolerogenization in T cells involved in the disorder. Methods oftreatment are known in the art. As discussed herein, one or more of thesubsets of DCs obtained by the methods described herein are suitable foruse in treatment of autoimmunity.

“Immunologic activity” of an antigen-binding fragment refers tospecifically binding the antigen which the intact antibody-recognizes.Such binding can or can not elicit an immune response. A specific immuneresponse can elicit antibody, B cell responses, T cell responses, anycombination thereof, and effector functions resulting therefrom.Included, without limitation, are the antibody-mediated functions ADCCand complement-mediated cytolysis (CDC). The T cell response includes,without limitation, T helper cell function, cytotoxic T cell function,inflammation/inducer T cell function, and T cell mediated immunesuppression. A compound (either alone or in combination with a carrieror adjuvant) able to elicit either directly or indirectly, a specificimmune response according to any of these criteria is referred to as“immunogenic.” Antigen-binding fragment “activity” or “function” refersto any of the immunologic activities of an antibody, includingdetection, amelioration or palliation of cancer.

An “immune response” refers to induction or enhancement of animmunologic response to malignant or diseased tissue, disease-causingagents and other foreign agents to which the body is exposed. Immuneresponses can be humoral, as evidenced by antibody production; and/orcell-mediated, as evidenced by cytolytic responses demonstrated by suchcells as natural killer cells or cytotoxic T lymphocytes (CTLs) and thecytokines produced thereby. Immune responses can be monitored by amononuclear cell infiltrate at the site of infection or malignancy.Typically, such monitoring is by histopathology. A “cancer-specificimmune response” is one that occurs against the malignancy but notagainst non-cancerous cells. The treatments described herein typicallyinduce or augment a cell-mediated immune response but can also induce oraugment an antibody-mediated immune response. The treatments can alsoinfluence the type of immune response to the antigen.

The compositions according to the invention are also suitable for use ininducing an antigen-specific Th1 immune response. Stimulating a Th1-typeimmune response can be measured in a host treated in accordance with theinvention and can be determined by any method known in the artincluding, but not limited to, a reduction in levels of IL-4 measuredbefore and after antigen challenge; or detection of lower (or evenabsent) levels of IL-4 in a treated host as compared to anantigen-primed, or primed and challenged, control treated without thecompositions of the invention; an increase in levels of IL-12, IL-18and/or IFN (α, β or γ, preferably IFN-γ in a treated host as compared toan antigen-primed or primed and challenged control; IgG2a antibodyproduction in a treated host as compared to an untreated control; areduction in levels of antigen-specific IgE as measured before and afterantigen challenge or detection of lower (or even absent) levels ofantigen-specific IgE in a treated host as compared to an antigen primedor primed and challenged untreated host. A variety of thesedeterminations can be made by measuring cytokines made by APCs and/orlymphocytes; preferably DCs and/or T cells, in vitro or ex vivo usingmethods described herein and known in the art. Methods to determineantibody production include any known in the art.

The Th1 biased cytokine induction produces enhanced cellular immuneresponses, such as those performed by NK cells, cytotoxic killer cells,Th1 helper and memory cells. These responses are particularly beneficialfor use in protective or therapeutic vaccination against viruses, fungi,protozoan parasites, bacteria, allergic diseases and asthma, as well astumors.

The invention further includes down-regulation of type I interferonproduction via ligation of BDCA-2, down-regulation of Th1 immuneresponses via ligation of BDCA-2, and polarization of an immune responseto Th2 via ligation of BDCA-2. These indications can be reversed byinterfering with ligation of BDCA-2. The invention further encompassesscreening for suitable moieties for interfering with ligation of BDCA-2and compositions of these moieties.

When antigen-binding fragments are used in combination with varioustherapeutic agents, the administration of both usually occurssubstantially contemporaneously. The term “substantiallycontemporaneously” means that they are administered reasonably closetogether with respect to time. The administration of the therapeuticagent can be daily, or at any other suitable interval, depending uponsuch factors, for example, as the nature of the ailment, the conditionof the patient and half-life of the agent.

Therapeutic compositions can be administered by injection or by gradualperfusion over time. The antigen-binding fragments can be administeredintravenously, intraperitoneally, intra-muscularly, subcutaneously,intracavity, intranodal, intrathecally or transdermally, alone or incombination with other therapeutic agents.

Another method of administration is intralesionally, for instance byinjection directly into the tumor. Intralesional administration ofvarious forms of immunotherapy to cancer patients does not cause thetoxicity seen with systemic administration of immunologic agents.Fletcher et al. (1987) Lymphokine Res. 6:45; Rabinowich et al. (1987)Cancer Res. 47:173; Rosenberg et al. (1989) Science 233:1318; and Pizzet al. (1984) J. Int. Cancer 34:359.

Further, it can be desirable to administer the compositions locally tothe area in need of treatment; this can be achieved by, for example,local infusion during surgery, by injection, by means of a catheter, orby means of an implant, the implant being of a porous, non-porous, orgelatinous material, including membranes, such as silastic membranes, orfibers. A suitable such membrane is Gliadel® provided by Guilfordsciences.

The fact that ligation of BDCA-2 with anti-BDCA-2 monoclonal antibody(AC144) induces intracellular Ca₂ ⁺ mobilization indicates thatplasmacytoid DC (and all other cells which express BDCA-2) can befunctionally modulated by triggering of BDCA-2 signaling or inhibitionof triggering of BDCA-2 signaling. Regarding functional modulation ofDC, the following aspects are encompassed by the

-   -   A) Induction and down-regulation of CD4⁺ and CD8⁺ T cells        responses.    -   B) Polarization of the immune response towards tolerance or        immunity    -   C) Polarization of CD4+ T cell responses towards Th1 cell        development, Th2 cells development or Th3/T-regulatory-1 CD4+ T        cell development. The latter down-regulate immune responses,        possibly via secretion of TGF-β and/or IL-10.    -   D) DC are usually thought of as antigen-presenting cells for T        cells. However, recent studies from several laboratories have        shown that they have important roles in B-cell activation and        regulation of antibody synthesis. B cell responses can therefore        be modulated via BDCA-2 on DCs. The same can also be true for NK        cell responses.

As type I interferon can induce Th1 type immune responses in humans(Parronchi et al. (1996) Eur. J. Immunol. 26:697-703), triggering ofBDCA-2 polarizes CD4⁺ T cell responses towards Th2 cell development,whereas inhibition of BDCA-2 signaling polarizes CD4⁺ T cell responsestowards Th1 cell development. The invention thus encompasses thepolarization of CD4⁺ T cell responses towards Th2 or Th1 celldevelopment by triggering of BDCA-2 signaling or inhibition oftriggering of BDCA-2 signaling, respectively.

All publications cited herein are hereby incorporated herein byreference in their entirety. The following examples are provided toillustrate, but not limit, the invention.

Example 1 Generation of DC-Specific mAB

Five 6-8 week old female Balb/c mice (Simonsen Laboratories, Gilroy,Calif.) were inoculated with approximately 5×10⁵ to 1×10⁶ purifiedHLA-DR⁺lin blood DC under anesthesia on d 0, 4, 7, 11, and 14 in theright hand footpad, and approximately 1×10⁶ HLA-A2⁺ Bristol-8 Blymphoblastoma cells in the left hand footpad on d −3, 0, 4, 7, 11, and14. Both cell types were incubated with 1:100 PHA (Gibco/BRL,Gaithersburg, Md.) for 10 min at room temperature and washed with PBSbefore injection. This treatment provides non-specific adjuvant effectsand obviates the need for adjuvants such as Freund's adjuvant.

On d 15, one day after the fifth injection of HLA-DR⁺ lin⁻ DC, the mouseright hand popliteal lymph nodes were removed. A lymphocyte suspensionwas prepared and the cells were fused to SP2/0 Ag14 myeloma cells usinga modification of the method described by Kohler and Milstein (1975)Nature 256:495. Fused cells were plated on 96-well plates in DMEMsupplemented with 20% FCS (HyClone, Logan, Utah), 2 mmol/L Lglutamine,15 mmol/L Hepes, 10⁻⁴ mmol/L hypoxanthine (Gibco/BRL), and placed in a37° C. incubator with 9% C0₂.

When visible hybridoma colonies were apparent, supernatants from thesewells were screened by flow cytometry for antibody secretion and fornon-reactivity (<1% positive cells) to PBMC. Briefly, a mixture of ratanti-mouse kappa mAb-conjugated polystyrene beads (2.5 μm in diameter,Interfacial Dynamics Corp., Portland, Oreg.) and PBMC was incubated with50 μl hybridoma supernatant for 20 min at room temperature. Thebead/cell mixture was then washed twice with PBS, pH 7.4, containing 5mmol/L EDTA and 0.5% BSA (PBS/EDTA/BSA), and binding of mouse IgM, IgG1,IgG2a and IgG2b from the supernatants to the beads and the test cellswas detected by staining with PE conjugated rat anti-mouse IgM mAb(clone X54, BD Biosciences, San Jose, Calif.), rat anti-mouse IgG1 mAb(clone X56, BD Biosciences) and rat anti-mouse IgG2 mAb (clone X57, BDBiosciences). PBMC and polystyrene beads can easily be discriminated inthe flow cytometric analysis by scatter signals.

Culture supernatants which fulfilled the screening criteria of the firstround were then screened by flow cytometric analysis for reactivity to asignificant proportion of blood DC. Briefly, a mixture of rat anti-mousemAb-conjugated polystyrene beads and enriched blood DC (PBMC depleted ofB cells, T cells and monocytes) was incubated with 50 μl hybridomasupernatant for 20 min at room temperature. The mixture was then washedtwice with PBS/EDTA/BSA, and stained with PE-conjugated rat anti-mouseIgM mAb, rat anti-mouse IgG1 mAb, and rat anti-mouse IgG2 mAb to detectbinding of mouse IgM, IgG1, IgG2a and IgG2b from the supernatants to thebeads and the enriched blood DC. For discrimination of HLA-DR⁺ DC fromHLA-DR⁻ cells in the flow cytometric analysis, the bead/cell mixture waswashed once, free binding sites of the PE-conjugated rat anti-mouse IgG2mAb and the bead-conjugated rat anti-mouse κ mAb were saturated byincubation with 100 μg/ml mouse IgG2a for 5 min at room temperature, andthe mixture was counter-stained with anti-HLA-DR-FITC (cloneAC122,IgG2a).

Selected hybridoma cells were expanded in culture, stocks were frozen inliquid nitrogen, subclones were established by limiting dilution, andseries of positive subclones were also frozen in liquid nitrogen. Theisotype of the mAb was determined by the ISOTYPE Ab-STAT Kit (SangStatMedical Corp., Palo Alto, Calif.).

For mAb production, hybridoma cells were either grown as an ascitestumor in Balb/c mice, with collection of mAb-rich ascites fluid, or incell culture (roller culture or hollow-fiber culture), with collectionof mAb-rich culture supernatant. Pure IgG mAb was prepared from ascitesfluid or cell culture supernatant by Protein A affinity chromatographyfollowed in some cases by hydrophobic interaction chromatography andstored in PBS with 5 mmol/L EDTA and 0.05% sodium azide at 4° C.Purified mAb were conjugated to FITC (Sigma, St. Louis, Mo.), PE(Cyanotech Corp., Kailua Kona, Hi.), Cy5 (Amersham Life Science Inc.,Arlington Heights, Ill.), APC (Europa Bioproducts Ltd., Cambridge, UK),biotin (Pierce, Rockford, Ill.) and colloidal super-paramagnetic beads(approximately 50 nm in diameter, Miltenyi Biotec GmbH, BergischGladbach, Germany) according to standard techniques. Hermanson (1996)Bioconjugate Techniques. Academic Press Inc., San Diego, 785 pp.; Aslamet al. (1998) Bioconjugation: protein coupling techniques for thebiomedical sciences. Macmillan Reference Ltd., London, 833 pp.; andKantor et al. (1997) Magnetic cell sorting with colloidalsuperparamagnetic particles. In, Cell Separation Methods andApplications. Recktenwald et al. Eds. Marcel Dekker Inc. New York, pp.153-173.

Cell Preparations

Buffy coats from normal healthy volunteers were obtained from theInstitute for Transfusionmedicine, Hospital Merheim, Cologne, Germany.PBMC were prepared from buffy coats by standard Ficoll-Paque (Pharmacia,Uppsala, Sweden) density gradient centrifugation.

Peripheral blood leukocytes were prepared from buffy coats by lysis oferythrocytes in isotonic ammonium chloride buffer (155 mmol/L NH₄Cl, 10mmol/L KHCO₃ and 0.1 mM EDTA). Hansel et al. (1991) J. Immunol. Met.145:105-110. CD4⁺ lin⁻ blood DC were isolated from PBMC by two-stepimmunomagnetic cell sorting (MACS) as described in detail elsewhere.Robert et al. (1999); and Miltenyi et al. (1999) High gradient magneticcell sorting. In, Flow cytometry and cell sorting. Ed., Radbruch.Springer-Verlag, Berlin. pp. 218-247. Briefly, monocytes, T cells, andNK cells were depleted using mAb against CD3 (Clone BW264/56), CD11b(clone M1/70.15.11.5), CD16 (Clone VEP-13) and in a few experiments apoorly defined antigen expressed on B cells and monocytes (clone L179).From the depleted cell fraction, blood DC were then enriched to highpurity using an antibody against CD4 (M-T321). To screen hybridomaculture supernatants (see above), blood DC were merely partiallyenriched by immunomagnetic depletion of T cells, B cells and monocytesbased on CD3 and L179 antigen expression.

CD1c⁻, BDCA-2⁻, and BDCA-3-expressing cells were isolated from PBMC ortonsils by indirect magnetic labeling with PE- or FITC-conjugated mAb(AD5-8E7, AC144 and AD6-5E8, respectively) as primary reagent andanti-PE or anti-FITC mAb-conjugated microbeads (Miltenyi Biotec GmbH) assecondary reagent, and enrichment of labeled cells by MACS. In someexperiments, BDCA-3⁺ cells were isolated based on direct magneticlabeling with anti-BDCA-3 mAb (AD5-5E8)-conjugated microbeads. Highlypure CD1c⁺ blood DC without contaminating CD1c⁺ B cells were obtained byimmunomagnetic depletion of CD18+ B cells using CD19 mAb-conjugatedmicrobeads (Miltenyi Biotec GmbH) followed by immunomagnetic enrichmentof CD11c cells. Basophils were purified from PBMC by immunomagneticdepletion of non-basophils based on indirect magnetic labeling of CD3-,CD7-, CD14-, CD15-, CD36-, CD45RA-, and HLA-DR-expressing cells with amagnetic labeling kit (Miltenyi Biotec). CD14⁺ monocytes, CD34⁺hematopoietic progenitor cells and CD3⁺ T cells were immunomagneticallypurified based on direct magnetic labeling with CD14, CD34 and CD3mAb-conjugated microbeads (Miltenyi Biotec GmbH), respectively.

Cell Culturing

For generation of “immature” monocyte-derived DC (Mo-DC), purified CD14⁺monocytes were cultured at a cell density of 5×10⁵ to 1×10⁶ cells/ml inmedium [RPMI 1640 (Gibco/BRL) supplemented with 2 mmol/L L-glutamine,10% FCS (Sigma), 100 mmol/L sodium pyruvate (Gibco/BRL), 100 U/mlpenicillin (Gibco/BRL), and 100 μg/ml streptomycin (Gibco/BRL)] at 37°C. in a humidified 5% CO₂-containing atmosphere in the presence of500-1000 U/ml rIL-4 (PeproTech, Rocky Hill, N.J.) and 100 ng/ml rGM-CSF(PeproTech) for 7 d. For generation of “mature” Mo-DC, “immature” Mo-DCwere washed once and cultured in medium in the presence of 20 ng/mlTNF-α (PeproTech) for another 3 d. For generation of CD34⁺ hematopoieticprogenitor cell-derived DC (CD34-DC), purified CD34+ cells were culturedat a cell density of 5×10⁴ cells/ml in medium in the presence of 100ng/ml rFlt3-Ligand (PeproTech), 0.5 ng/mL rTGF-β1 (PeproTech), 10 ng/mlrTNF-α, 20 ng/ml rSCF (PeproTech) and 100 ng/ml rGM-CSF for 11 d.Freshly isolated CD4⁺lin⁻ blood DC were cultured at a cell density of5×10⁵ to 1×10⁶ cells/ml in medium in the presence of 10 ng/ml rIL-3(PeproTech) for up to 48 h. Isolated CD1c−, BDCA-2−, and BDCA-3−expressing DC were cultured at a cell density of 5×10⁵ to 1×10⁶ cells/mlin medium without any cytokines or in the presence of 10 ng/ml rIL-3, 20ng/ml IL-4 (PeproTech) and 100 ng/ml GM-CSF for up to 48 h.

Example 2

Flow Cytometric Analysis of Blood DCs

A FACScalibur (BD Biosciences) was used for one-, two-, three- orfour-color flow cytometry. Data of 5×10³ to 2×10⁵ cells per sample wereacquired in list mode and analyzed using CellQuest software (BDBiosciences).

The following mAb (clone names) were used in this study for flowcytometry: CD1a (HI149), CD10 (HI10a), CD11a (G43-25B), CD11c (B-1y6),CD25 (M-A261), CD27 (M-T271), CD32 (FL18.26), CD38 (HIT2), CD40 (5C3),CD43 (IG10), CD54 (HA58), CD62L (Dreg 56), CD64 (10.1), CD69 (FN50),CD98 (UM7F8), anti-HLA-DQ (TU169), and anti-TCRαβ T10B9.1A-31 fromPharMingen, San Diego, Calif.; CD2 (S5.2), CD8 (SK1), CD13 (L138), CD14(MFP9), CD19 (SJ25-C1), CD33 (P67.6), CD34 (8G12), CD45RO (UCHL-1), CD56(NCAM16.2), CD71 (L01.1), CD123 (9F5), anti-IgD (TA4.1), anti-mouse IgG1(X56), anti-mouse IgG2 (X57), and anti-mouse IgM (X54) from BDBiosciences; CD5 (CLB-T11/11, 6G4), CD7 (CLB-T-3A1/1, 7F3), CD16(CLB-FcR gran/1, 5D2), CD45RA (F8-11-13), CD80 (CLB-DALI) from CLB,Amsterdam, Netherlands; CD18 (7E4), CD23 (9P25); CD58 (AICD58), CD77(38.13), CD83 (HB15A), CD86 (HA5.2B7), CD116 (SC06) from CoulterImmunotech, Marseilles, France; CD4 (M-T321), CD11b (M1/70.15.11.5),CD14 (TÜK4), CD15 (VIMC6), anti-HLA-DR (910/D7), anti-AC133 (AC133/1),and anti-TCRαβ (BW242/412) from Miltenyi Biotec GmbH, CD36 (AC106),CD123 (AC145), anti-HLA-DR (AC122 and AC123) and anti-GPA (AC107) fromAmcell, Sunnyvale, Calif.; CD1c (M241) from Ancell, Bayport, Minn.;polyclonal anti-IgG, anti-IgM (SA-DA4), polyclonal anti-kappa, andpolyclonal anti-lambda from Southern Biotechnology Associates,Birmingham, Ala.; CD61 (VIPL2) from W. Knapp, Institute of Immunology,University of Vienna, Vienna, Austria; CD44 (IM7) from J. Moll,Forschungszentrum Karlsruhe, Karlsruhe, Germany; CD20 (H147) from CaltagLaboratories, Burlingame, Calif.; anti-CLA (HECA-452) from E. Butcher,Department of Pathology, Stanford University, Stanford, Calif.;anti-Fc_(ε)RI (15-1) from J. P. Kinet, Molecular Allergy and ImmunologySection, National Institute of Allergy and Infectious Diseases, NationalInstitutes of Health, Rockville, Md.; CD11c (Ki-M1) from M. R.Parwaresch, Department of Pathology, Christian Albrechts University,Kiel, Germany; CMRF-44 and CMRF-56 from D. N. Hart, Mater MedicalResearch Institute, Mater Misericordiae Hospitals, South Brisbane,Queensland, Australia; and anti-HLA-A, -B, -C (W6/32) from Sigma.

All antibodies were used as FITC-, PE-, biotin- or Cy5-conjugated mAb.For indirect immunofluorescent staining with biotinylated mAb,streptavidin-APC (BD Biosciences) was used. To exclude dead cells in theflow cytometric analysis, cells were stained with propidium iodide. Tominimize Fc receptor-mediated mAb binding, cells were stained in mostexperiments in the presence of FcR-blocking reagent (Miltenyi BiotecGmbH) containing human IgG.

Microscopic Analysis

Cells were spun down on slides in a cytocentrifuge (Cytospin 3, Shandon,Pittsburgh, Pa.). For fluorescence microscopy, slides were air driedovernight after cytocentrifugation and mounted with Fluoromount G(Southern Biotechnology Associates). For May Grunwald/Giemsa staining,slides were air dried for at least 2 h after cytocentrifugation, stainedin May Grunwald/Giemsa solution (Merck, Darmstadt, Germany) for 2 min atroom temperature, rinsed thoroughly in distilled water, stained inGiemsa solution (Merck) for 15 min at room temperature, washedrepeatedly in distilled water, and air dried for at least 2 h. A ZeissAxioscop, microscope (Zeiss, Oberkochen, Germany) was used for analysis.Digital pictures were made using the Xillix Microlmager M11400-12X(Xillix, Vancouver, Canada).

Example 3 Cross-Inhibition, Co-Capping and Co-Internalization Analysis

To analyze whether two different mAb clones recognize the same (or aclosely related) antigen epitope, cross-inhibition binding assays wereperformed. Between 1×10⁶ and 2×10⁶ cells were pre-incubated with one ofthe two mAb clones at a concentration of about 100 μg/ml for 10 min at4° C., and then stained with a PE-conjugate of the other mAb clone atits optimal titer for another 5 min at 4° C. PBMC were used to analyzecross-inhibition of BDCA-2-, BDCA-3-, and BDCA-4-specific mAb clones andMOLT-4 cells were used to analyze cross-inhibition of CD1c-specific mAbclones. Cell staining was analyzed by flow cytometry.

To ascertain whether AD5-5E8 and AD5-14H12 recognize the same antigen(or the same antigen-complex) a co-capping assay was performed. Briefly,BDCA-3-expressing cells were isolated from PBMC by indirect magneticlabeling with PE-conjugated AD5-14HI2 mAb and anti-PE mAb-conjugatedmicrobeads, and isolated cells were incubated for 30 min at 37° C. toinduce capping of the mAb-antigen complex. Afterwards, cells were washedwith ice cold PBS/EDTA/BSA supplemented with 0.1% sodium azide(PBS/EDTA/BSA/azide), and stained with FITC-conjugated AD5-5E8 mAb inPBS/EDTA/BSA/azide for 10 min at 4° C. Cell staining was analyzed byfluorescence microscopy.

A co-internalization assay was used to investigate whether AC144 andAD5-17F6 recognize the same antigen (or the same antigen-complex).Briefly, 1×10⁶ PBMC were incubated with 50 μg/ml AC144 mAb for 15 min atroom temperature in PBS/BSA, washed twice in PBS/BSA, and then incubatedin cell culture medium at 37° C. for 30 min. To analyze whether AC144mAb is internalized upon culturing, aliquots of the cells were stainedbefore and after the culture period with rat anti-mouse IgG1-PE. Todetermine whether all AC144 mAb-binding sites were saturated withunconjugated AC144 mAb before culturing and whether any free bindingsites reappear after culturing, aliquots of the cells were stainedbefore and after the culture period with AC144-PE. To analyze whetherAD5-17F6 antigen is co-internalized, aliquots were stained before andafter the culture period with AD5-17F6-PE. All cells were counterstained with CD123-FITC and HLA-DR-Cy5 to be able to gate onCD123^(bright)HLA-DR⁺ plasmacytoid DC in the flow cytometric analysis.

Example 4 Endocytosis Assay

To assess endocytosis of blood DC subsets, purified CD1c⁺, BDCA-2⁺ andBDCA-3⁺ blood DC, and (as control) purified CD3⁺ T cells were incubatedat 37° C. in medium with 1 mg/ml Lucifer yellow (LY) for 0, 15, 45, and75 min. Afterwards, cells were washed three times in ice coldPBS/EDTA/BSA and analyzed by flow cytometry.

Example 5 Reactivity of Isolated Blood DCs with Non-Cultured Blood Cells

According to their reactivity with blood cells, the mAb listed in Table1 could be divided into four groups: (1) AC144, AD5-13A11 and ADB-4B8;(2) AD5-17F6; (3) AD5-5E8 and AD5-14H12; and (4) AD5-8E7.

The mAb of the first group, AC144, AD5-13A 1 and ADB-4B8, stainapproximately 0.41±0.17% (n=10) of all PBMC (FIG. 1A). In a dot plot offorward and side scatter signals, these rare cells constitute ahomogeneous cell population that is located between small restinglymphocytes and monocytes (FIG. 1B). Accordingly, these rare cells donot express the αβ T cell receptor (TCRαβ), CD14, CD19 and CD56 (FIG.1A), lineage markers which are expressed on T cells, monocytes, B cellsand NK cells, respectively. Staining of highly purified blood DC (>95%HLA-DR⁺, TCRαβ⁻, CD14⁻, CD19⁻ and CD56⁻) reveals that the mAb of thefirst group are reactive with CD11c⁻CD123^(bright) blood DC (FIG. 2) butnot reactive with CD11c⁺ blood DC. To analyze whether all of them reactwith a single antigen, we performed two-color stainings andcross-inhibition studies. The results show that all mAb of this grouprecognize a single epitope of the same antigen. This antigen was namedBDCA-2.

As shown in FIG. 3, the mAb of the second group, AD5-17F6, recognizesthe same cells among PBMC as AC144, one of the BDCA-2-specific mAb ofthe first group. Nevertheless, AD5-17F6 stains an antigen which isdifferent from BDCA-2. This was unequivocally demonstrated byco-internalization experiments, where AD5-17F6 showed surface stainingwith equal intensity before and after anti-BDCA-2 mAb-mediatedinternalization of BDCA-2, and by staining of DC after culture, whereAC144 mAb and AD5-17F6 mAb showed entirely different staining patterns(FIG. 4). The antigen recognized by AD5-17F6 was named BDCA-4 and isidentical to neuropilin-1. He et al. (1997).

The mAb of the third group, AD5-5E8 and AD5-14H12, stain approximately0.04±0.01% (n=10) of all PBMC (FIG. 1A). According to scatter signals(FIG. 1B) and counterstaining with mAb against the TCRαβ, CD14, CD19 andCD56 (FIG. 1A), these cells are distinct from lymphocytes and monocytesand slightly larger than the cells recognized by the antibodies of thefirst group. Accordingly, staining of blood DC shows that a differentsubset is recognized by AD5-5E8 and AD5-14H12, namely CD11c^(dim)CD123⁻blood DC (FIG. 2). According to two-color stainings, cross-blockingstudies and co-capping experiments both mAb appear to recognize twospatially unrelated epitopes of the same antigen. We named this antigenBDCA-3.

The fourth group, mAb AD5-8E7, reacts with up to 2.39±0.96% (n=10) ofunfractionated PBMC (FIG. 1A). Light-scatter analysis (FIG. 1B) andcounter-staining of the lineage markers TCRαβ, CD14, and CD19 revealthat the mAb is not reactive to T cells and monocytes, but is reactiveto a major subset of small resting CD19⁺ B cells. Staining of purifiedDC shows that AD5-8E7, in addition to B cells, stains a third subset ofblood DC distinct from those subsets recognized by the mAb of the firstand the second group, namely CD11c^(bright)CD123^(dim) blood DC. Asignificant proportion of the CD11C^(bright)CD123^(dim) blood DCexpresses CD56 (see below). For this reason, some AD5-8E7-reactive PBMCstain for CD56 (FIG. 1A). AD5-8E7 is not reactive to purified NK cells.The antigen recognized by AD5-8E7 was initially named BDCA-1 as itappeared to be a new antigen. However, it later transpired that AD5-8E7completely blocks binding of the CD1c mAb M241 to MOLT-4 cells (FIG. 6).Thus, the antigen recognized by AD5-8E7 is CD1c.

None of the mAb listed in Table 1 is reactive with granulocytes,platelets, erythrocytes, purified basophils and purified CD34⁺hematopoietic progenitor cells.

Example 6 Expression of BDCA-2, BDCA-3 and BDCA-4 on Cultured Blood DC,Mo-DC and CD34-DC

Freshly isolated plasmacytoid CD11c⁻ blood DC depend on IL-3 forsurvival and maturation, whereas survival and maturation of CD11c⁺ bloodDC is far less cytokine-dependent. Expression of BDCA-2, BDCA-3 andBDCA-4 on CD11c⁻ and CD11c⁺ blood DC was analyzed after 0 h, 1 h, 3 h, 6h, 9 h, 12 h, 18 h, 24 h, 36 h, and 48 h of culture of total blood DC inthe presence of rIL-3. The results are shown in FIG. 4. Expression ofBDCA-2 is completely down-regulated within 48 h on CD11c⁻ blood DC. Incontrast, BDCA-4 is even further up-regulated on CD11c⁻ blood DC and,unlike BDCA-2, is also expressed to a high level on most, if not all,CD11c⁺ DC. Expression of BDCA-3 is rapidly induced on CD11c⁻ blood DC,reaching the highest expression level after 24 h. Thereafter, BDCA-3expression appears to be down-regulated again. Analyzing the expressionof BDCA-3 on CD11c⁺ blood DC is complicated by the fact thatBDCA-3⁻CD11c^(bright) and BDCA-3⁺CD11c^(dim) subsets are present at theonset of the culture. Expression of BDCA-3 remains unchanged at leastuntil 6 h of culture on the BDCA-3⁺CD11c^(dim) blood DC population, andis induced within 3 h on at least some cells of theBDCA-3-CD11c^(bright) blood DC subset. Expression of BDCA-2, BDCA-3 andBDCA-4 on Mo-DC and CD34⁻ DC

Functional CD1a⁺ DC were generated ex vivo from monocytes and from CD34⁺hematopoietic progenitor cells. Bender et al. (1996); Pickl et al.(1996; Romani et al. (1994); Sallusto et al. (1994); Caux et al. (1992);Mackensen et al. (1995); Szabolcs et al. (1995); Herbst et al. (1996);de Wynter et al. (1998); and Strunk et al. (1996). FIG. 7 shows thatMo-DC, which were generated by culturing monocytes for 7 d in thepresence of rGM-CSF and IL-4 and CD34-DC, generated by culturing CD34+hematopoietic progenitor cells for 11 d in the presence of rFlt3-Ligand,rTGF-β1, rTNF-α, rSCF and rGM-CSF, express CD1a, CD1c and BDCA-4, butneither BDCA-2 nor BDCA-3.

Example 7 Internalization of BDCA-2 Upon Anti-BDCA-2 mAB-MediatedCross-Linking

The possibility that 37° C. incubation of anti-DCA-2 mAb-labeled BDCA-2⁺cells results in mAb internalization was addressed by staining of PBMCwith FITC-conjugated AC144 mAb (IgG1). Then, following incubation at 37°C., remaining cell surface associated mAb was detected by staining withPE-conjugated rat anti-mouse IgG1 mAb. As shown in FIG. 8, when cellswere incubated at 37° C., the intensity of the rat anti-mouse IgG1-PEstaining decreases extremely rapidly to background levels. In contrast,the intensity of the AC144-FITC staining decreases only temporarily to alevel of approximately 50%, but thereafter nearly returns to thepre-incubation level. This demonstrates that BDCA-2 is internalized uponanti-BDCA-2 mAb cross-linking, with kinetics similar toreceptor-mediated endocytosis. The transient decrease in AC144-FITCstaining intensity is probably due to patching and capping of theBDCA-2/anti-BDCA-2 mAb complex before endocytosis.

Example 8 Morphology of Isolated CD1c⁺, BDCA-2⁺ and BDCA-3⁺ Blood DC

CD1c⁺, BDCA-2⁺ and BDCA-3⁺ cells were isolated from PBMC by indirectmagnetic labeling with PE-conjugated primary mAb and anti-PEAb-conjugated microbeads and enrichment of labeled cells by MACS (FIG.9). On May Grunwald/Giemsa staining of cytocentrifuge slides (FIG. 9),freshly isolated BDCA-2-expressing cells display the typicallymphoplasmacytoid morphology of CD11c⁻ CD4⁺lin⁻ DC from blood andtonsils: that is, medium-sized round cells with oval or indented nuclei.In contrast, both freshly isolated CD1c⁺ blood DC as well as freshlyisolated BDCA-3⁺ blood DC display the typical morphologicalcharacteristics of CD11c⁺CD4⁺ lin⁻ DC from blood or tonsils: that is,less rounded cells with short cell processes and more hyperlobulatednuclei. In addition to CD1c⁺ BDC, CD1c⁺ B cells with the typicalmorphology of small resting lymphocytes can be seen on thecytocentrifuge slides of isolated CD1c⁺ PBMC. Highly pure CD1c⁺ BDC areobtained if, prior to the enrichment of CD1c⁺ cells, CD19⁺ B cells aremagnetically depleted from PBMC.

Example 9 Surface Phenotype of CD1c+, BDCA-2⁺ and BDCA-3⁺ Blood DC

The phenotype of BDCA-2⁺ and BDCA-3⁺ blood DC was analyzed by two-colorimmunofluorescence with PE- and FITC-conjugated mAb. For analysis ofCD1c⁺ blood DC, three-color stainings were performed using CD19-Cy5 forexclusion of B cells. The results of the phenotypic analysis are shownin Table 2 and can be summarized as follows: none of the blood DCsubsets express CD1a, CD8, CD15, CD16, CD19, CD20, CD23, CD25, CD27,CD34, CD61, CD69, CD7.1, CD77, CD80, CD83, glycophorin A (GPA), TCRαβ,AC133, IgD, IgM and the CMRF-56 antigen. All blood DC subsets expressCD43, CD44, CD54 and MHC class I molecules at a similar level. BDCA-2⁺blood DC differ from the other two subsets in that they do not expressCD13, CD40; CD45RO, CD56, but CD45RA and little amounts of CD10, and inthat they express lower levels of CD18, CD38, CD58, CD98, CD116 and CLA,but higher levels of CD4. CD1c⁺ blood DC differ from the other twosubsets in that they express CD2, higher levels of MHC class11molecules, but lower levels of CD62L, and in that they express the Fcreceptors CD32, CD64 and Fc_(ε)R1. Probably due to the Fcreceptor-expression, CD1c⁺ blood DC are also positive for IgG, kappa andlambda. Furthermore, some CD1c⁺DC are positive for CD14 and CD11b,whereby the level of expression inversely correlates with the level ofboth CD1c and CD2 expression. BDCA-3⁺ blood DC differ from the other twosubsets in that they express CD36 at a much lower level and in that theyappear to express low levels of CD5. Finally, apart from CD11c andCD123, at least one additional antigen, CD33, is useful fordiscrimination of all three subsets: CD33 is expressed at low levels onBDCA-2⁺ DC, at intermediate levels on BDCA-3⁺ DC and at high levels onCD1c⁺ DC.

TABLE 2 Antigen Clone BDCA-2⁺ BDCA-3⁺ CD1c⁺ CD1a HI149 ⁻ ⁻ ⁻ CD1c M241 ⁻⁻ + CD2 S5.2 ⁻/minor ⁻ + subset+ CD4 M-T321 ++ + + CD5 CLB-T1/1, 6G4 ⁻⁻/+ ⁻ CD7 CLB-T3A1, 7F3 ⁻/minor ⁻ + subset+ CD8 SK1 ⁻ ⁻ ⁻ CD10 HI1Oa ⁻/+⁻ ⁻ CD11a G43-25B + ++ + CD11b M1/7O.15.11.5 ⁻ ⁻ ⁻/+ CD11c Ki-M1 ⁻ + ++CD13 L138 ⁻ + + CD14 TUK4 ⁻ ⁻ ⁻/+ CD15 VIMC6 ⁻ ⁻ ⁻ CD16 CLB-FcR Gran/1 ⁻⁻ ⁻ CD18 7E4 + ++ ++ CD19 SJ25-C1 ⁻ ⁻ ⁻ CD20 HI47 ⁻ ⁻ ⁻ CD23 9P25 ⁻ ⁻ ⁻CD25 M-A251 ⁻ ⁻ ⁻ CD27 M-T271 ⁻ ⁻ ⁻ CD32 FL18.26 (2003) ⁻ ⁻ + CD33 P67.6⁻/+ + ++ CD34 8G12 ⁻ ⁻ ⁻ CD36 AC106 + ⁻/+ + CD38 HIT2 + ++ ++ CD40 FC3 ⁻⁻/+ ⁻/+ CD43 1G10 + + + CD44 IM7 + + + CD45RA F8-11-13 + ⁻ ⁻ CD45ROUCHL-1 ⁻ + + CD54 HA58 + + + CD56 NCAM16.2 ⁻ ⁻/subset+ ⁻/subset+ CD58AICD58 + ++ ++ CD61 VIPL2 ⁻ ⁻ ⁻ CD62L DREG56 ⁻ + + CD64 10.1 ++ ++ +CD69 FN50 ⁻ ⁻ ⁻ CD71 LO1.1 ⁻ ⁻ ⁻ CD77 38.13 ⁻ ⁻ ⁻ CD80 DAL-1 ⁻ ⁻ ⁻ CD83HB15A ⁻ ⁻ ⁻ CD86 HA5.2B7 + ++ +++ CD98 HIM6 ++ +++ +++ CD116 SC06 + ++++ CD123 AC145 ++ ⁻ + HLA-DR AC122 + + ++ HLA-DQ TU169 + + ++ HLA-A, B,C W6/32 + + + GPA AC107 ⁻ ⁻ ⁻ TCRαβ T10B9.1A-31 ⁻ ⁻ ⁻ AC133 AC133 ⁻ ⁻ ⁻Fc_(ε)R I 15-1 ⁻ ⁻ + IgD TA4.1 ⁻ ⁻ ⁻ IgG Polyclonal ⁻ ⁻ + IgM SA-DA4 ⁻ ⁻⁻ Kappa Polyclonal ⁻ ⁻ + Lambda Polyclonal ⁻ ⁻ + CLA HECA-452 ++ +++ +++CMRF44 CMRF44 ⁻ ⁻ ⁻/minor subset+ CMRF56 CMRF56 ⁻ ⁻ ⁻

Example 10 Expression of MHC Class II, CD83 and Co-Stimulatory Moleculeson CD1c⁺, BDCA-2⁺ and BDCA-3⁺ Blood DC after Culture

Freshly isolated CD1c⁺ blood DC and BDCA-3⁺ blood DC were cultured for 1d in medium without any supplemented cytokines and freshly isolatedBDCA-2⁺ blood DC were cultured for 2 d in medium supplemented with IL-3and CD40 mAb on CD32-transfected fibroblasts. After the culture period,cells were analyzed for the expression of CD1a, CD80, CD83, CD86 andHLA-DR. For comparison, so-called “immature” Mo-DC, generated byculturing of monocytes for 7 d in the presence of GM-CSF and IL-4, andso-called “mature” Mo-DC, generated by culturing of “immature” Mo-DC for3 d in the presence of TNF-α, were also included. Sallusto et al. (1995)J. Exp. Med. 182:389-400; and Sallusto et al. (1998) J. Immunol.28:2760-2769. As shown in FIG. 10, in contrast to “immature” Mo-DC and“mature” Mo-DC, none of the blood DC subsets expresses CD1a after theculture period. However, the costimulatory molecules CD80 and CD86, theDC activation antigen CD83 (Zhou et al. (1995); Zhou et al. (1992) J.Immunol. 149:735-742; and Zhou et al. (1996) Proc. Natl. Acad. Sci. USA93:2588-2592), and HLA-DR molecules are up-regulated upon culturing onall three blood DC subsets to a similar level as compared to matureMo-DC. The results were not significantly different in anotherexperiment in which all three blood DC subsets were cultured for 2 d inmedium supplemented with IL-3, IL-4 and GM-CSF. As has been previouslyshown for CD11c⁻CD4⁺lin⁻ DC from blood and tonsils, BDCA-2⁺ blood DCrapidly die when cultured in medium without IL-3.

Example 11 Endocytic Capacity of Freshly Isolated CD1c⁺, BDCA-2⁺ andBDCA-3⁺ Blood DC

The endocytic capacity of purified CD1c⁺, BDCA-2⁺ and BDCA-3⁺ blood DC,and, as a control, of purified CD3⁺ T cells was examined by culturingthe cells at 37° C. in the presence of LY and analyzing the uptake of LYafter various periods of time by flow cytometry. As shown in FIG. 11,unlike purified CD3⁺ T cells, purified CD1c⁺ blood DC, BDCA-3⁺ blood DC,and to some extent also BDCA-2⁺ blood DC have the ability to endocytoseLY. Similar results were obtained using FITC-Dextran. The endocyticcapacities of all blood DC populations are much lower if compared withMo-DC.

The amino acid sequence of BDCA-4 was obtained by purifying the antigenwith AD5-17F6 mAb (AD5-17F6 affinity column) and analyzing the purifiedantigen by MALDI TOF mass spectrometry. BDCA-4 is identical toneuropilin-1. He et al. (1997).

Example 12 Ligation of BDCA-2 with Anti-BDCA-2 Monoclonal Antibody(AC144) Induces Intracellular Ca²⁺ Mobilization, Whereas Ligation ofBDCA-4 (Neuropilin-1) with Anti-BDCA-4 does not Induce Ca²⁺ Mobilization

Materials and Methods:

Measurement of cytosolic calcium in BDCA-2⁺ BDCA-4⁺ BDC andBDCA-2-transfected or non-transfected U937 cells. BDCA-2⁺BDCA-4⁺ bloodDC and BDCA-2-transfected or non-transfected U937 cells were loaded withIndo-1 AM (Sigma, St. Louis, Mo.) as described by Valitutti et al.(1993) Eur. J. Immunol. 23:790-795. Anti-BDCA-2 (AC144, IgG1) oranti-BDCA-4 (AD5-17F6, IgG1) mAb were added to freshly isolatedBDCA-2⁺BDCA-4⁺ BDC and BDCA-2-transfected or non-transfected U937 cells,respectively, followed or not followed by rat anti-mouse IgG1 mAb (X56)as cross-linker Cells were analyzed on a flow cytofluorimeter to detectCa²⁺ fluxes. Only live (based on scatter criteria) and Indo-1-labeledcells (based on 405 nm versus 525 nm emission spectra) were included inthe analysis.

FIG. 13 shows intracellular mobilization is induced inimmunomagnetically purified BDCA-2⁺BDCA-4+ BDC (A, B) andBDCA-2-transfected U937 cells (D), but not in non-transfected U937 cells(E) via anti-BDCA-2 mAb alone (A) and or anti-BDCA-2 plus crosslinkingsecondary mAb (B, D, E).

Ligation of BDCA-4 on immunomagnetically purified BDCA-2⁺BDCA-4⁺ BDCwith anti-BDCA-4 mAb and cross-linking secondary mAb does not inducecytosolic Ca²⁺-mobilization. Shown is the Ca²⁺-dependent 405 nm/525 nmratio of Indo-1-fluorescence (Y-axis) against time (X-axis, a value of1024 corresponds to 204.80 sec).

As shown in FIG. 13, ligation of surface BDCA-2 on plasmacytoid BDC(FIGS. 13A and B) and BDCA-2-transfected U937 cells (FIG. 13D) with aspecific mAb (AC144, IgG1) followed (FIGS. 13B and D) or not followed(FIG. 13A) by a secondary cross-linking mAb (rat anti-mouse IgG1, X56)elicited a rapid and transient rise in cytosolic calcium concentration.On the contrary, incubation of plasmacytoid DC with anti-BDCA-4 mAb(AD5-17F6) followed by a secondary cross-linking mAb (rat anti-mouseIgG1, X56) (FIG. 14C), or of non-transfected U937 cells with anti-BDCA-2mAb (AC144, IgG1) followed by a secondary cross-linking mAb (ratanti-mouse IgG1, X56) (FIG. 13E) did not induce a rapid and transientrise in cytosolic calcium concentration.

Example 13 Production of Type I Interferon by Purified BDCA-2⁺ BDCA-4⁺BDC in Response to Viral Stimulation (Influenza Virus Strain PR8) isInhibited by Triggering of BDCA-2 with Anti-BDCA-2 mAB

CD4⁺CD123^(bright)CD11c⁻ plasmacytoid DC were shown to be the chief typeI interferon producers in response to enveloped viruses, bacteria, andtumor cells. Fitgerald-Bocarsly et al. (1993) Pharmacol. Ther. 60:39-62;Siegal et al. (1999) Science 284:1835-1837; Cella et al. (1999) NatureMed. 5:919-923. For this reason, they have also been called natural typeI interferon producing cells (NIPC). Plasmacytoid DC express BDCA-2 andBDCA-4. As shown in FIG. 14, ligation of surface BDCA-2 on plasmacytoidDC with a specific mAb followed by a secondary cross-linking mAb (ratanti-mouse IgG1, X56), inhibits secretion of type I interferon byimmunomagnetically purified plasmacytoid BDCA-2⁺BDCA-4⁺ DC from blood ortonsils in response to stimulation with influenza virus strain PR8 (5HAU/ml). The level of type I interferon production in cultures withanti-BDCA-2, influenza virus and cross-linking mAb (FIG. 14,AC144+RamG1+FLU) is much lower as in cultures with influenza virus alone(FIG. 14, FLU), or with an isotype control mAb (anti-cytokeratin mAbCK3-11D5, IgG1), influenza virus and cross-linking mAb (FIG. 14,CK3+RamG1+FLU).

Conversely, ligation of surface BDCA-4 on plasmacytoid DC with aspecific mAb followed by a secondary cross-linking mAb (rat anti-mouseIgG1, X56), does not inhibit secretion of type I interferon byimmunomagnetically purified plasmacytoid BDCA-2⁺BDCA-4⁺ DC from blood ortonsils in response to stimulation with influenza virus strain PR8 (5HAU/ml). The level of type I interferon production in cultures withanti-BDCA-4, influenza virus and cross-linking mAb (FIG. 14,17F6+RamG1+FLU) is the same as in cultures with an isotype control mAb(anti-cytokeratin mAb CK3-11D5, IgG1), influenza virus and cross-linkingmAb (FIG. 14, CK3+RamG1+FLU).

Materials and Methods:

BDCA-2- and BDCA-4-expressing plasmacytoid DC were isolated from PBMC(FIG. 14A) or tonsillar cells (FIG. 14B) by direct magnetic labelingwith anti-BDCA-4 (AD5-17F6)-conjugated microbeads and enrichment oflabeled cells by MACS. Isolated BDCA-2- and BDCA-4-expressingplasmacytoid DC were cultured for 24 hours in medium in the presence of:a) IL-3 alone (FIG. 14, Control); b) IL-3, anti-BDCA-2 mAb (AC144, IgG1)and rat anti-mouse IgG1 mAb (FIG. 14, AC144+RamG1); c) IL-3, anti-BDCA-2mAb (AC144, IgG1), rat anti-mouse IgG1 mAb, and influenza virus strainPR8 (FIG. 14, AC144+RamG1+FLU); d) IL-3 and influenza virus strain PR8(FIG. 14, FLU); e) IL-3, anti-cytokeratin mAb (CK3-11D5, IgG1), ratanti-mouse IgG1 mAb, and influenza virus strain PR8 (FIG. 14,CK3+RamG1+FLU); and f) IL-3, anti-BDCA-4 mAb (AD5-17F6), rat anti-mouseIgG1 mAb, and influenza virus strain PR8 (FIG. 14 17F6+RamG1+FLU).

Secreted type I interferon in the culture supernatants was measured byevaluating inhibition of Daudi cell proliferation (Nederman et al.(1990) Biologicals 18:29-34) with reference to a standard IFN-α curve.

Regarding the inhibition of type I interferon production byBDCA-2⁺BDCA-4⁺ plasmacytoid DC, increased levels of circulating type Iinterferon and of type I interferon inducing factor (something like acomplex of anti-DNA antibody and DNA) are found in SLE patients andcorrelate to disease activity. Furthermore, patients with non-autoimmunedisorders treated with type I interferon frequently developautoantibodies and occasionally SLE. Several papers from Ronnblom et al.(1999) Clin. Exp. Immunol. 115: 196-202; (1999) J. Immunol. 163:6306-6313; and (2000) J. Immunol. 165: 3519-3526) show that type Iinterferon inducing factors derived from patients induce secretion oftype I interferon in PBMC from healthy donors and they selectivelyactivate natural type I interferon producing cells (NIPC=plasmacytoidDC).

The findings presented herein that ligation of BDCA-2 suppresses theproduction of type I interferon induced by viral stimulation show thatbinding to BDCA-2 can be applied to treat the disease not just byligation of BDCA-2 but also by depleting NIPC (=BDCA-2+ BDCA-4+plasmacytoid DC). The invention thus further encompasses in vivo, invitro and ex vivo depletion of NIPC. Such depletion is suitable for usein treatment or prophylaxis of autoimmune diseases.

FIG. 14 shows ligation of BDCA-2 but not of BDCA-4 with a specific mAbfollowed by a secondary cross-linking mAb inhibits secretion of type Iinterferon by plasmacytoid BDCA-2⁺BDCA-4⁺ DC from blood or tonsils inresponse to stimulation with influenza virus strain PR8. PlasmacytoidBDCA-2⁺BDCA-4⁺ DC from blood (A) or tonsils (B) were cultured for 24hours in the presence of IL-3 alone (control); IL-3, anti-BDCA-2 mAb andrat anti-mouse IgG1 mAb (AC144+RamG1); IL-3, anti-BDCA-2 mAb, ratanti-mouse IgG1 mAb, and influenza virus strain PR8 (AC144+RamG1+FLU);IL-3 and influenza virus strain PR8 (FLU); IL-3, anti-cytokeratin mAb,rat-anti-mouse IgG1 mAb, and influenza virus strain PR8 (CK3+RamG1+FLU);IL-3, anti-BDCA-4 mAb, rat anti-mouse IgG1 mAb, and influenza virusstrain PR8 (17F6+RamG1+FLU). Secreted type I interferon (U/ml) in theculture supernatants was measured by a bioassay with reference to astandard type I interferon curve.

Example 14

BDCA-2 is not Only Able to Endocytose a Ligand, but Also to Deliver itto an Antigen-Processing and Loading Compartment, and to Present it toCD⁴⁺ Class II-Restricted T Cells

Materials and Methods:

BDCA-2- and BDCA-4-expressing plasmacytoid DC were isolated from PBMC bydirect magnetic labeling with anti-BDCA-4 (AD5-17F6)-conjugatedmicrobeads and enrichment of labeled cells by MACS. Isolated BDCA-2- andBDCA-4-expressing plasmacytoid DC were co-cultured with 4×10⁴ cells/wellof the B13 T cell clone (Lanzavecchia et al. (1988) J. Exp. Med.167:345-352) in 96-well flat-bottom microplates in the presence of IgG1mAbs (0.2 μg/ml). mAbs used in the assay were the following: AC144(anti-BDCA-2, IgG1), ZM3.8 (anti-ILT3, IgG1) and CK3-11D5(anti-cytokeratin, IgG1). After 48 hours, the cultures were pulsed with(³H)thymidine (1 μCi/well), and the radioactivity incorporated wasmeasured after additional 16 hours. (3H) Thymidine uptake (cpm) wasplotted against the number of isolated BDCA-2- and BDCA-4-expressingplasmacytoid DC in the cultures (FIG. 15).

FIG. 15 shows presentation of anti-BDCA-2 mAb (AC144, IgG1) to a T cellclone specific for mouse IgG1 by isolated BDCA-2- and BDCA-4-expressingplasmacytoid DC. BDCA-2⁺BDCA-4⁺ plasmacytoid DC present anti-BDCA-2 mAb(AC144, IgG1, ▪) to T cells much more efficiently than anti-ILT-3 mAb(ZM3.8, IgG1, ▴) and anti-cytokeratin mAb (CK3-11D5, IgG1, ).

Incubation of anti-BDCA-2 mAb (AC144, IgG1)-labeled BDCA-2⁺BDCA-4⁺plasmacytoid DC at 37° C. results in extremely rapid internalization ofthe anti-BDCA-2 mAb/BDCA-2 complexes on the cell surface (see FIG. 8).Here, it is shown that the anti-BDCA-2 mAb (AC144, IgG1) accesses anantigen-processing and loading compartment and peptides derived from theantibody are efficiently presented to a CD4⁺ class II-restricted T cellclone (B13) specific for a mouse IgG1 peptide epitope. The presentationof the anti-BDCA-2 mAb was compared to that of an IgG1 mAb that binds toa receptor (ILT3) known to be capable of targeting its ligand(s) intoprocessing and peptide-loading compartments, and to that of an IgG1 mAbthat does not bind to a cell-surface molecule on BDCA-2⁺BDCA-4⁺plasmacytoid DC (anti-cytokeratin mAb CK3-11D5, IgG1), but can be takenup in the fluid phase. As shown in FIG. 15, BDCA-2⁺BDCA-4⁺ plasmacytoidDC presented anti-BDCA-2 mAb (AC144) to T cells much more efficientlythan the anti-ILT-3 mAb and the anti-cytokeratin mAb.

Example 15 In Tonsillar Cells, Expression of BDCA-2 is Restricted toCD123⁺ T Cell-Zone Associated Plasmacytoid DC, Whereas BDCA-4 May Alsobe Expressed at Low Levels on a Few Other Cells

FIG. 16 shows expression of BDCA-2 and BDCA-4 on tonsillar plasmacytoidCD123⁺ DC. Shown are two-color stainings of tonsillar cells with aFITC-conjugated mAb against BDCA-2 (AC144) and a PE-conjugated mAbagainst CD123 and BDCA-4 (AD5-17F6), respectively. Note that expressionof BDCA-2 is restricted to CD123bright plasmacytoid DC, whereas BDCA-4is also expressed at low levels on a few other cells.

Example 16 BDCA-4 mAB (AD5-17F6) Recognizes Neuropilin-1

Neuropilin-1 is a receptor for the collapsin/semaphorin family thatmediates neuronal cell guidance. Neuropilin-1 is also expressed byendothelial and tumor cells as an isoform-specific receptor for vascularendothelial growth factor. However, it was not known before, thatneuropilin-1 is expressed on plasmacytoid DC in blood and tonsils andthat it represents an excellent marker for plasmacytoid DC at least infresh non-cultured blood.

Material and Methods:

Neuropilin-1 was immunoprecipitated from cell lysates of non-transfectedPAE cells (P) and neuropilin-1-transfected PEA cells (NP) (Soker et al.(1998) Cell 92:735-745) using the anti-BDCA-4 mAb AD5-17F6 (anti-NRP-1(ML)). Precipitated proteins were analyzed by SDS-PAGE and Westernblotting with the BDCA-4-specific mAb AD5-17F6 (ML) or aneuropilin-1-specific mAb from Shay Soker, Children's Hospital, Boston,Mass. (S).

FIG. 17 shows that neuropilin-1 was immunoprecipitated from cell lysatesof neuropilin-1-transfected PEA cells (NP) but not of non-transfectedPAE cells (P) with the anti-BDCA-4 mAb AD5-17F6 (anti-NRP-1 (ML)).Precipitated proteins were analyzed by SDS-PAGE and Western blottingwith the BDCA-4-specific mAb AD5-17F6 (ML) or a neuropilin-1-specificmAb from Shay Soker, Children's Hospital, Boston, Mass. (S).

Note that the BDCA-4-specific mAb AD5-17F6 immunoprecipitates a specificband of about 130-140 kDa from neuropilin-1-transfected PEA cells (NP),but not from non-transfected PAE cells (P). The band can be detectedwith the neuropilin-1-specific mAb from Shay Soker (S) but not with theanti-BDCA-4 mAb AD5-17F6 (ML). Thus, our anti-BDCA-4 mAb AD5-17F6recognizes the native form of neuropilin-1 in standardimmunoprecipitation experiments, but fails to detect the denatured formof neuropilin-1 when used in SDS-PAGE/Western blotting experiments.

Interestingly, neuropilin-1 is also expressed by endothelial and tumorcells as an isoform-specific receptor for vascular endothelial growthfactor (VEGF). More interestingly, several papers (Gabrilovich et al.(1996) Nature Med. 2:1267; Nature Med. 2: 1096-103; Gabrilovich et al.(1999) Clin. Cancer Res. 5: 2963-70; Ohm et al. (1999). J. Immunol. 163:3260-8; Oyama et al. (1998) J. Immunol. 160:1224-32; Gabrilovich et al.(1998). Blood 92:4150-66; Ishida et al. (1998) J. Immunol. 161:4842-51)have shown that VEGF produced by a large percentage of tumors decreasesDC generation and function in vivo. It is not clear whether theseeffects on DCs are mediated by neuropilin-1 (BDCA-4), but the inventionencompasses neuropilin-1-mediated functional modulation of DCs.

Example 17 Production of Type I Interferon (IFN-α) by Purified BDCA-2BDCA-4⁺ BDC in Response to Stimulation with Poly I:C is Inhibited byTriggering of BDCA-2 with Anti-BDCA-2 mAB

CD4⁺CD123^(bright)CD11c⁻ plasmacytoid DC were shown to be the chief typeI interferon producers in response to enveloped viruses, bacteria, andtumor cells. Fitzgerald-Bocarsly et al. (1993) Pharmacol. Ther.60:39-62; Siegal et al. (1999) Science 284: 1835-1837; Cella et al.(1999) Nature Med. 5:919-923. For this reason, they have also beencalled natural type I interferon producing cells (NIPC). Plasmacytoid DCexpress BDCA-2 and BDCA-4. As shown in FIG. 19, ligation of surfaceBDCA-2 on plasmacytoid DC with a specific mAb followed by a secondarycross-linking mAb (goat anti-mouse IgG), inhibits secretion of IFN-α byimmunomagnetically purified plasmacytoid BDCA-2⁺BDCA-4⁺ DC from blood ortonsils in response to stimulation with polyI:C. The level of IFN-αproduction in cultures with anti-BDCA-2, poly I:C and cross-linking mAb(FIG. 18, AC144+Goat anti-mouse IgG+Poly I:C) is lower as in cultureswith mouse IgG1, poly I:C and cross-linking mAb (FIG. 18, MouseIgG1+Goat anti-mouse IgG+Poly I:C).

Materials and Methods:

CD11c⁻ CD123^(bright) plasmacytoid DC were separated from humanperipheral blood mononuclear cells using BDCA-4 microbeads. CD11c⁻C123^(bright) plasmacytoid DC (1×10⁶ cells/ml) were incubated with 10μg/ml of AC144 mAb or mouse IgG1 mAb (CF6B, anti-TPO) in RPMI, 10% FCS,10 mM HEPES, 50 μM 2-ME, 20 μg/ml gentamicin at 37° C. for 30 min. 20μg/ml of goat anti-mouse IgG (Chemicon International) were added andcells were again incubated at 37° C. for 30 min. These cells werecultured with or without 20 μg of poly I:C (Sigma) at 37° C. for 24hours. Culture supernatants were harvested and IFN-α concentrations weredetermined by ELISA (Endogen). The sensitivity of the assay is 3 pg/ml.

FIG. 18 shows ligation of BDCA-2 but not of BDCA-4 with a specific mAbfollowed by a secondary cross-linking mAb inhibits secretion of IFN-α byplasmacytoid BDCA-2⁺BDCA-4⁺ DC from blood or tonsils in response tostimulation with poly I:C. Plasmacytoid BDCA-2+ BDCA-4+ DC from bloodwere cultured with 10 μg/ml of AC144 mAb (2 and 4) or mouse IgG1 mAb(CF6B, anti-TPO, 1 and 3) at 37° C. for 30 min. 20 μg/ml of goatanti-mouse IgG were added and the cells were again incubated at 37° C.for 30 min. These cells were cultured with (3 and 4) or without (1 and2) 20 μg of poly I:C at 37° C. for 24 hours. Culture supernatants wereharvested and IFN-α concentrations were determined by ELISA.

Example 18 BDCA-2 mRNA Expression Analysis by RT-PCR in Various Tissuesand Purified Blood Cell Populations

Nucleic and Amino Acid Sequences

The cDNA encoding BDCA-2 was obtained by expression cloning in COScells. FIG. 5 shows the amino acid sequence of BDCA-2 (the isoform withall six exons expressed). BDCA-2 is a novel C-type lectin type IImembrane protein. Such lectins are described, for instance in Bates etal. (1999) J. Immunol. 163:1973-1983. Comparison of BDCA-2 to knownC-type lectins is shown in Example 20.

FIG. 19 shows on analysis of human multiple tissue cDNA panels fromClonetech (lane 1: heart; lane 2: brain; lane 3: placenta; lane 4: lung;lane 5: liver; lane 6: skeletal muscle; lane 7: kidney; lane 8:pancreas; lane 9: spleen; lane 10: thymus; lane 11: testis; lane 12:ovary; lane 13: small intestine; lane 14: lymph node; lane 15: bonemarrow; lane 16: fetal liver; lane 17: tonsil) and on analysis of cDNAsprepared from different populations of blood leukocytes (lane 18: Tcells; lane 19: B cells; lane 20: NK cells; lane 21: monocytes; lane 22:CD11c^(bright)CD123^(low)DC; lane23: CD11c-CD123^(bright) plasmacytoidBDC) for the presence of BDCA-2 cDNA.

All cDNAs were normalized to the mRNA expression level of severaldifferent housekeeping genes (glyceraldehyde-3-phosphate dehydrogenase,phospholipase A2, α-tubulin, and β-actin). Normalization ensures anaccurate assessment of tissue specificity and relative abundance oftarget mRNAs. The same amount of cDNA (about 50 pg) was used for eachRT-PCR reaction. RT-PCR reactions were performed with specific primersfor BDCA-2 (forward: 5′-TTGAAAGAACCACACCCCGAAAGT (SEQ ID NO:7) andreverse: 5′-TAGCTTTCTACAACGGTGGATGCC (SEQ ID NO:8)) and primers for thefour housekeeping genes (CLONTECH) mentioned above using AdvanTaq PlusDNA Polymerase (CLONTECH).

Cycle conditions were as follows: 94° C. for 30 sec and 68° C. for 2min. 34 cycles were used for BDCA-2 and 38 cycles forglyceraldehyde-3-phosphate dehydrogenase (G3PDH). Note that BDCA-2 mRNAsignals are only detected in CD 11c⁻CD123^(bright) plasmacytoid DC. Iffour more PCR cycles were used for amplification of BDCA-2 cDNA (38cycles instead of 34 cycles), weak signals were also detected inpancreas, testis, ovary, bone marrow and tonsil. With cDNA from testis(38 PCR cycles), signals of shorter transcripts (splice variants) weremore prominent as compared to the signals from CD11c⁻ CD123^(bright)plasmacytoid DC and signals from the full-length transcript wereactually only detectable with even more PCR cycles.

Example 19

Exon/Intron Structure of BDCA-2 and Splice Variants of BDCA-2

The information on the splice variants of BDCA-2 was obtained by RT-PCRamplification of mRNA from plasmacytoid DC with primers complementary tomRNA sequences in front of the start codon (forward primer:5′-TTGAAAGAACCACACCCCGAAAGT (SEQ ID NO:7)) and behind the stop codon(reverse primer: 5′-TAGCTTTCTACAACGGTGGATGCC(SEQ ID NO:8)), cloning ofthe resulting fragments in plasmids and sequencing of the inserts. Theresults are shown in FIG. 20. For comparison, splice variants of mousedendritic cell-associated C-type lectin 2 (Dectin-2) are shown in FIG.21.

FIG. 22 shows an alignment of the mRNA sequences of BDCA-2 and mouseDectin-2 with the positions of the deduced introns being indicated.Table 3 shows the parameters of the exons.

TABLE 3 amino acid Number of residues amino acid Exon mRNA encodedresidues encoded 0  0-361 0 1 362-522  1-10 10 2 523-615 11-41 31 3616-726 42-78 37 4 727-872  78-127 49 5 873-988 128-166 39 6  989-1283167-213 47

The positions of the introns are based on Homo sapiens Chromosome 12Clone RP11-277J24, Working Draft Sequence, 21 unordered pieces (GenBankAccession Number AC006517) and the rules for splicing of transcripts.The intron/exon makeup of BDCA-2 is similar to that of Dectin-2.

At least four splice variants of BDCA-2 are produced. These are an mRNAencoding a protein with all six exons; an mRNA encoding a proteincontaining exons 1, 3, 4, 5, and 6; an mRNA encoding a proteincontaining exons 1, 2, 4, 5, and 6 and an mRNA encoding a proteincontaining exons 1, 2, 3, 5, and 6.

Example 20 BDCA-2 Homology and Protein Domains

An alignment of the amino acid sequences of human BDCA-2, human DCIR(dendritic cell immunoreceptor), and mouse Dectin-2 (dendriticcell-associated C-type lectin-2) is shown in FIG. 23.

Human DCIR (GenBank Accession Number AJ133532) is the molecule with thehighest homology to BDCA-2 among human molecules (see Bates et al.(1999) J. Immunol. 163:1973) with about 51% of the aa being identicalover a stretch of 191 aa.

Mouse Dectin-2 (GenBank Accession Number AF240357) is most likely themurine homolog of human BDCA-2 (see Ariizumi et al. (2000) J. Biol.Chem. 16:11957; WO 98/28332; PCT/US97/23761; and U.S. Pat. No.6,046,158) with about 51% of the aa being identical over a stretch of211 aa.

BDCA-2 (213 aa), DCIR (237 aa) and Dectin-2 (209 aa) are all type IImembrane glycoproteins of the calcium-dependent (C-type) lectin family.Each of the molecules contains a putative cytoplasmic domain (BDCA-2: aa1-21; DCIR: aa 1-44; Dectin-2: aa 1-17), a putative transmembrane domain(BDCA-2: aa 22-41; DCIR: 44-69; Dectin-2: 18-40), and a putativeextracellular domain (BDCA-2: aa 42-213; DCIR: 70-237; Dectin-2:40-209). Within the putative extracellular domain, each of the moleculescontains a single carbohydrate recognition domain (CRD) at theCOOH-terminal end (BDCA-2: aa 83-206; DCIR: 106-230; Dectin-2: 79-202).FIG. 23 shows the alignment of human BDCA-2, human DCIR and mouseDectin-2.

Putative protein domains/motifs as found using the PROSITE database areshown in Table 4.

TABLE 4 Domain BDCA-2 ASN glycosylation 110-113 NCSV (SEQ ID NO: 9)137-140 NSSY (SEQ ID NO: 10) 164-167 NVTF (SEQ. ID NO: 11)cAMP- and cGMP- dependent protein  53-56 KRLS (SEQ ID NO: 14)kinase phosphorylation site 135-138 SQKProtein Kinase C phosphorylation site  51-53 TVK 107-109 SQKCasein kinase II phosphorylation site 123-126 TREE (SEQ ID NO: 16)187-190 SSEE (SEQ ID. NO: 17) Tyrosine kinase phosphorylation site 57-64 KLREYQQY (SEQ ID NO: 30) Amidation site148-151 GGRR (SEQ ID NO: 32) N-myristylation siteC-type lectin domain signature 180-206 Dectin-2 ASN glycosylation131-134 NESL (SEQ ID NO: 12) cAMP- and cGMP- dependent proteinkinase phosphorylation site Protein Kinase C phosphorylation site 15-17 TLR  49-51 SRR  72-74 SEK  94-96 STKCasein kinase II phosphorylation site  94-97 STKE (SEQ ID NO: 18)101-104 STSE (SEQ ID NO: 19) 119-122 TEAE (SEQ ID NO: 20)200-203 SICE (SEQ ID NO: 21) Tyrosine kinase phosphorylation site 50-58 RRLYELHTY (SEQ ID NO: 31) Amidation site N-myristylation site 11-16 GVCWTL (SEQ ID NO: 33)  68-73 GTMVSE (SEQ ID NO: 34) 77-82 GCCPNH 9SEQ ID NO: 35) C-type lectin domain signature 11-17 176-202 DCIR ASN glycosylation 185-188 NESS (SEQ ID NO: 13)CamP- and cGMP- dependent protein  78-81 KKTT (SEQ ID NO: 15)kinase phosphorylation site Protein Kinase C phosphorylation site 80-82 TTK 130-132 SEK 211-213 SPK Casein kinase II phosphorylation site  1-9 TYAE (SEQ ID NO: 22)  80-83 TTKE (SEQ ID NO: 23) 87-90 TTLE (SEQ ID NO: 24) 126-129 SWQD (SEQ ID NO: 25)130-133 SEKD (SEQ ID NO: 26) 146-149 TQEE (SEQ ID NO: 27)168-171 SDPE (SEQ ID NO: 28) 228-231 SVCE (SEQ ID NO: 29)Tyrosine kinase phosphorylation site Amidation site N-myristylation site 20-25 GINTAS (SEQ ID NO: 36) C-type lectin domain signature 203-230

BDCA-2 contains three putative N-glycosylation sites (aa 110-113 NCSV;aa 137-140 NSSY; aa 164-167 NVTF), whereas Dectin-2 (aa 131-134 NESL)and DCIR (aa 185-188 NESS) contain only one putative N-glycosylationsite. All the putative phosphorylation sites of BDCA-2 and Dectin-2 arelocated in the putative extracellular domain. Thus, it is ratherunlikely that they become phosphorylated by intracellular kinases. Likemany C-type lectins (e.g. CD94, Ly-49, and NKG2) that are encoded in thenatural killer gene complex, DCIR contains the consensus immunoreceptortyrosine-based inhibitory motif (ITIM motif; (I/V)XYXX(L/V) (SEQ IDNO:37)) in the cytoplasmic domain (aa 5-10 ITY AEV (SEQ ID NO:38)).Interestingly, this ITIM motif is not found in the relatively shortcytoplasmic tail of BDCA-2 and Dectin-2 (BDCA-2: 21 aa; Dectin-2: 17aa).

Example 21 BDCA-3 Protein Analysis

BDCA-3-expressing HD-MY-Z cells were stimulated for 24 hours with 10ng/ml PMA (Sigma) and 0.5 mg/ml Ionomycin to up-regulateBDCA-3-expression. 3×10⁷ PMA/Ionomycin stimulated HD-MY-Z cells weresurface biotinylated by incubation for 15 minutes at 4° C. with 1 mg/mlSulfo-NHS-LC-Biotin (Pierce), and washed twice. Cells were resuspendedin 50 mM Tris-HCl pH 8.0 supplemented with 10% sucrose and proteinaseinhibitors (Phenylmethylsulfonylfluoride, Pepstatin A, Leupeptin, andAprotinin from Serva) and at 0° C. ultrasonified (5×4 seconds, 70%output). Sonified cells were centrifuged at 900×g at 4° C. for 10minutes to remove nuclei and intact cells. The supernatant wascentrifuged at 30,000×g at 4° C. for 2 hours to obtain purified cellmembranes. Membranes were lysed by incubation in 50 mM Tris-HCl pH 8.0,150 mM NaCl supplemented with proteinase inhibitors and 1% NP-40 for 1hour at 0° C. Non-solubilized membrane fragments were removed bycentrifugation at 30,000×g at 4° C. To the supernatant, MnCl₂ and CaCl₂were added to a final concentration of 1 mM each. The lysate wasadsorbed onto a ConA Sepharose column (1 ml), and bound proteins wereeluted with 10 ml elution buffer (0.5 M D(+) Mannose, 20 mM Tris-HCl pH7.4, 0.5 M NaCl, 1% NP-40) and concentrated to a volume of 1 ml usingCetriprep-10 centrifugal concentrators (Amicon).

The proteins were pre-cleared by incubation with 150 μl anti-NIPmAb-conjugated MicroBeads (Miltenyi Biotec) for 30 minutes at 4° C. andμMACS column separation. For specific immunoprecipitation of BDCA-3,proteins were either incubated with 2 μg of the NIP-conjugatedBDCA-3-specific mAb AD5-14H12 (IgG1) or for control of specificity with2 μg of the NIP-conjugated CD19-specific mAb SJ25-C1 (IgG1) as primaryreagent for 14 hours at 4° C., and with anti-NIP mAb-conjugatedMicroBeads as secondary reagent for 3 hours at 4° C. Precipitatedproteins were isolated by MACS column separation. Retained proteins wereeluted with 70 μl SDS-PAGE buffer containing DTT. Precipitated proteinswere analyzed by SDS-PAGE (4-12%) and Western blotting withstreptavidin-peroxidase.

The results in FIG. 24 show that the BDCA-3-specific mAb AD5-14H12specifically immunoprecipitates a cell surface protein of about 100 kDfrom HD-MY-Z cells. Thus, BDCA-3 has an apparent molecular weight of 100kD.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity andunderstanding, it will be apparent to those skilled in the art thatcertain changes and modifications can be practiced. Therefore, thedescription and examples should not be construed as limiting the scopeof the invention, which is delineated by the appended claims.

1. An ex vivo or in vitro cell population comprising dendritic cellslabeled with an antibody or an antigen-binding fragment that is specificfor BDCA-1.
 2. The cell population of claim 1, wherein said antibody orantigen-binding fragment constitutes one or more of the following: abispecific antibody; a chimeric antibody; a murine antibody; a humanantibody; a humanized antibody a monoclonal antibody; a hybrid antibody;a recombinant antibody; an Fab fragment; a F(ab′)₂ fragment; a Fab′fragment; a single chain V region fragment (ScFv); an aptamer; anisolated H and L chain; or a fusion polypeptide.
 3. The cell populationof claim 1, wherein said antibody or antigen-binding fragment is fusedto a chemical functional moiety.
 4. The cell population of claim 3,wherein the chemical functional moiety is a signal peptide, an agentthat enhances immunologic reactivity, an antigen, an agent thatfacilitates coupling to a solid support, a bioresponse modifier, animmunotoxin or a toxin.
 5. The cell population of claim 3, wherein thechemical functional moiety is biotin or avidin.
 6. The cell populationof claim 3, wherein the chemical functional moiety is a cytokine orchemokine.
 7. The cell population of claim 6, wherein the cytokine orchemokine is IL-1, IL-2, IL-4, IL-5, IL-6, IL-12, an interferon, TNF-α,IL-10 or TGF-β.
 8. The cell population of claim 3, wherein the chemicalfunctional moiety is a toxin selected from ricin, a radionuclide,pokeweed antiviral protein, Pseudomonas exotoxin A, diphtheria toxin,ricin A chain, a fungal ribosome inactivating protein, and aphopholipase enzyme.
 9. The cell population of claim 1, wherein theantibody or antigen-binding fragment is conjugated to a detectable labelor a paramagnetic label.
 10. The cell population of claim 9, wherein thedetectable label is a radioisotope, an enzyme, a fluorescent compound, achemi-luminescent compound, a bio-luminescent compound, a substrateco-factor, or an inhibitor.
 11. The cell population of claim 1, whereinthe antibody or antigen-binding fragment is conjugated to asuper-paramagnetic bead.
 12. A pharmaceutical composition comprising acell population according to claim 1, together with a physiologicallyacceptable excipient.
 13. A method for enriching for dendritic cellsfrom a mixture of human cells comprising: (a) contacting cells in themixture of human cells with an antibody or antigen-binding fragmentspecific for BDCA-1 under conditions where the antibody orantigen-binding fragment labels BDCA-1 positive cells; and (b)separating dendritic cells labeled by the antibody or antigen-bindingfragment from other cells in the mixture.
 14. The method of claim 13,wherein the mixture of human cells is blood or a cell populationobtained from blood, wherein the mixture is substantially free of tumorcells expressing BDCA-1.
 15. The method of claim 13, wherein thedendritic cells are separated by immunomagnetic cell sorting (MACS). 16.The method of claim 13, wherein the dendritic cells are separated byflow cytometry (FACS).
 17. The method of claim 13, wherein the mixtureof human cells is obtained from bone marrow, peripheral blood or fromcell culture.
 18. The method of claim 13, wherein the mixture of humancells is obtained by leukopheresis of peripheral blood.
 19. The methodof claim 13, wherein the mixture of human cells is obtained fromumbilical cord blood, tonsil, lymph node, nasal membrane, spleen, skin,airway epithelia, lung, liver, gut, thymus or Peyers patches.
 20. Amethod of modulating dendritic cell cytokine production, comprising: a)obtaining a substantially pure population of dendritic cells accordingto the method of claim 14; and then b) treating the population with anagent that modulates dendritic cell cytokine production.
 21. A method ofmodulating the immune capacity of dendritic cells, comprising: a)obtaining a substantially pure population of dendritic cells accordingto the method of claim 14; and then b) modulating calcium mobilizationof dendritic cells in the population.
 22. A method of modulatingdendritic cell cytokine production, comprising: a) obtaining an enrichedpopulation of dendritic cells according to the method of claim 13; andthen b) treating the population with an agent that modulates dendriticcell cytokine production.
 23. A method of modulating the immune capacityof dendritic cells, comprising: a) obtaining an enriched population ofdendritic cells according to the method of claim 13; and then b)modulating calcium mobilization of dendritic cells in the population.24. The method of claim 13, which is a method for detecting,enumerating, or isolating dendritic cells.
 25. The method of claim 13,wherein the mixture of human cells is substantially free of tumor cells,endothelial cells and neuronal cells.